METHODS OF TREATING CANCER USING MULTI-SPECIFIC BINDING PROTEINS THAT BIND NKG2D, CD16 AND A TUMOR-ASSOCIATED ANTIGEN CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/156,214, filed March 3, 2021, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 28, 2022, is named DFY-103WO_SL.txt and is 194,985 bytes in size. FIELD OF THE DISCLOSURE [0003] The present disclosure relates to methods of treating cancer using multi-specific binding proteins that bind NKG2D, CD16 and a tumor-associated antigen such as HER2. In one aspect, the multi-specific binding protein is used in combination with a corticosteroid to reduce the risk of infusion-related reactions. In another aspect, the multi-specific binding protein is used to treat cancer that has low or moderate HER2 expression levels. The present disclosure also relates to pharmaceutical formulations containing the multi-specific binding proteins. BACKGROUND [0004] Cancer continues to be a significant health problem despite the substantial research efforts and scientific advances reported in the literature for treating this disease. Cancer immunotherapies are being developed to facilitate destruction of cancer cells using the patient’s own immune system. The immune cells activated by cancer immunotherapies include T cells and natural killer (NK) cells. For example, bispecific T-cell engagers are designed to direct T cells against tumor cells, thereby rendering cytotoxicity against the tumor cells. Bispecific antibodies that bind NK cells and a tumor-associated antigen (TAA) have also been created for cancer treatment (see, e.g., WO 2016/134371). [0005] HER2 is a transmembrane glycoprotein in the epidermal growth factor receptor family. It is a receptor tyrosine kinase and regulates cell survival, proliferation, and growth.
HER2 plays an important role in human malignancies. The ERBB2 gene is amplified or overexpressed in approximately 30% of human breast cancers. Patients with HER2- overexpressing breast cancer have substantially lower overall survival rates and shorter disease-free intervals than patients whose cancer does not overexpress HER2. Moreover, overexpression of HER2 leads to increased breast cancer metastasis. Over-expression of HER2 is also known to occur in many other cancer types, including ovarian, esophageal, bladder and gastric cancer, salivary duct carcinoma, adenocarcinoma of the lung, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. [0006] Multi-specific binding proteins that bind HER2 and one or more immune cell surface proteins have been studied. For example, WO 2018/152518 describes multi-specific binding proteins that bind HER2, NKG2D, and CD16. The present disclosure adds to these developments and provides clinical methods, including dosage regimens, to treat patients with specific HER2-targeting cancer immunotherapies with desired safety and efficacy. Furthermore, the present disclosure adds to the earlier developments in the field by providing formulations containing such cancer immunotherapies that are sufficiently stable and suitable for administration to patients. SUMMARY OF THE DISCLOSURE [0007] The present disclosure, in various embodiments, provides methods of treating cancer using a multi-specific binding protein having an antigen-binding site that binds a tumor-associated antigen such as HER2, an antigen-binding site that binds NKG2D, and an antibody Fc domain. The multi-specific binding protein can be used in combination with a corticosteroid to reduce the risk of infusion-related reactions. Alternatively, the multi- specific binding protein can be formulated at a high concentration, as disclosed herein, into a small volume, thereby allowing subcutaneous administration and reducing the risk of infusion-related reactions. The multi-specific binding protein can also be used to treat cancer that has low or moderate HER2 expression levels. [0008] Accordingly, in one aspect, provided herein is a method of treating cancer by administering to a subject in need thereof a therapeutically effective amount of a multi- specific binding protein and a therapeutically effective amount of a corticosteroid to reduce infusion related reactions to the multi-specific binding protein. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen binding site that binds a tumor-associated antigen; and (c) a third antigen binding site that
binds CD16. In some embodiments, the infusion related reactions include cytokine release syndrome, anaphylaxis, chills, fever/pyrexia, hypotension, hypertension, rigors, headache, dizziness, itching, sore throat, laryngeal edema, angioedema, redness/flushing, rash/urticaria, bronchospasm, tachycardia, bradycardia, auricular fibrillation, hypoxia, respiratory distress/dyspnea/shortness of breath/breathless sensation, chest tightness, nausea, vomiting, pain (e.g., chest pain, back pain), shivering, tremors, myalgia, tiredness, insomnia, asthenia, hypersensitivity, and/or diarrhea. In some embodiments, the corticosteroid is a glucocorticoid. In some embodiments, the glucocorticoid is selected from methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, or pharmaceutically acceptable salts of any thereof. In particular embodiments, the glucocorticoid is methylprednisolone. In some embodiments the effective amount of methylprednisolone is 125 mg. In other particular embodiments, the glucocorticoid is dexamethasone. [0009] In some embodiments, the corticosteroid is administered parenterally. In some embodiments the corticosteroid is administered intravenously. In some embodiments the corticosteroid is administered orally. In some embodiments the corticosteroid is administered prior to the administration of the multi-specific binding protein. In some embodiments the corticosteroid is administered within 6 hours prior to the administration of the multi-specific binding protein. In some embodiments, the corticosteroid is administered within 1 hour prior to the administration of the multi-specific binding protein. [0010] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of an antihistamine. In some embodiments the antihistamine is diphenhydramine. In some embodiments, the therapeutically effective amount of diphenhydramine is 40-50 mg. In some embodiments, the antihistamine is administered intravenously. In some embodiments, the antihistamine is administered within 60 minutes or within 30 minutes prior to the administration of the multi-specific binding protein. [0011] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of an antipyretic. In some embodiments, the antipyretic is acetaminophen. In some embodiments, the therapeutically effective amount of acetaminophen is 800-1000 mg. In some embodiments, the antipyretic is administered
intravenously. In some embodiments, the antipyretic is administered within 60 minutes or within 30 minutes prior to the administration of the multi-specific binding protein. [0012] In some embodiments, the tumor-associated antigen is HER2. In some embodiments, the cancer has HER2 expression level scored as 1+ or 2+. [0013] In another aspect, the present disclosure provides a method of treating a cancer having a HER2 expression level scored as 1+ by administering a therapeutically effective amount of a multi-specific binding protein to a subject in need thereof. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0014] In another aspect, the present disclosure provides a method of treating a cancer having a HER2 expression level scored as 2+ by administering a therapeutically effective amount of a multi-specific binding protein to a subject in need thereof. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0015] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a locally advanced or metastatic solid tumor. In some embodiments, the cancer is selected from breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, non-small cell lung cancer (NSCLC), glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, or gallbladder cancer. In particular embodiments, the cancer is urothelial bladder cancer or metastatic breast cancer. [0016] In some embodiments, the multi-specific binding protein is administered to the subject in an initial four-week treatment cycle on day 1, day 8, and day 15. In some embodiments, the multi-specific binding protein is not administered on day 22. In some embodiments, the method further includes administering the multi-specific binding protein to the subject on day 1 and day 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. In some embodiments, each of the doses includes the multi-specific binding protein at an amount selected from 5.2 × 10
-5 mg/kg, 1.6 × 10
-4 mg/kg,
5.2 × 10
-4 mg/kg, 1.6 × 10
-3 mg/kg, 5.2 × 10
-3 mg/kg, 1.6 × 10
-2 mg/kg, 5.2 × 10
-2 mg/kg, 1.6 × 10
-1 mg/kg, 0.52 mg/kg, 1.0 mg/kg, 1.6 mg/kg, 5.2 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg. In some embodiments, the multi-specific binding protein is administered by intravenous infusion. In some embodiments, the multi-specific binding protein is administered subcutaneously. [0017] In some embodiments, the multi-specific binding protein is used as a monotherapy. In some embodiments, the method further includes administering to the subject an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, 200 mg of pembrolizumab is administered on day 1 of the initial treatment cycle. In some embodiments, if the subject receives one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles. [0018] In some embodiments, a corticosteroid is administered to the subject on day 1 of the initial four-week cycle. In some embodiments, an antihistamine is administered to the subject on day 1 of the initial four-week cycle. In some embodiments, an analgesic is administered to the subject on day 1 of the initial four-week cycle. [0019] In some embodiments, the multi-specific binding protein is formulated in a pharmaceutical composition that includes histidine, a sugar or sugar alcohol, and a polysorbate, at pH 5.5 to 6.5. In some embodiments, the pharmaceutical composition includes greater than 50 mg/mL of the multi-specific binding protein. [0020] In another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof a pharmaceutical composition that includes: (i) greater than 50 mg/mL of a multi-specific binding protein incorporating (a) a first antigen- binding site that binds NKG2D, (b) a second antigen-binding site that binds a tumor- associated antigen, and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; (ii) histidine; (iii) a sugar or sugar alcohol; and (iv) a polysorbate, at pH 5.5 to 6.5. [0021] In another aspect, the present disclosure provides a method of treating cancer having a HER2 expression level scored as 1+ by administering to a subject in need thereof: (i) a pharmaceutical composition that includes: greater than 50 mg/mL of a multi-specific binding protein, histidine, a sugar or sugar alcohol, and a polysorbate, at pH 5.5 to 6.5, and (ii) a therapeutically effective amount of a corticosteroid to reduce one or more infusion-
related reactions to the multi-specific binding protein.. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0022] In another aspect, the present disclosure provides a method of treating cancer having a HER2 expression level scored as 2+ by administering to a subject in need thereof: (i) a pharmaceutical composition containing greater than 50 mg/mL of a multi-specific binding protein, histidine, a sugar or sugar alcohol, and a polysorbate, at pH 5.5 to 6.5; and (ii) a therapeutically effective amount of a corticosteroid to reduce one or more infusion- related reactions to the multi-specific binding protein. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0023] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a locally advanced or metastatic solid tumor. In certain embodiments, the cancer is breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, non-small cell lung cancer (NSCLC), glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, or gallbladder cancer. In certain embodiments, the cancer is urothelial bladder cancer or metastatic breast cancer. [0024] In certain embodiments, the pharmaceutical composition is administered to the subject in an initial four-week treatment cycle on day 1, day 8, and day 15. In certain embodiments, the pharmaceutical composition is not administered on day 22. In certain embodiments, the method further includes administering the pharmaceutical composition to the subject on day 1 and day 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. [0025] In certain embodiments, each of the administered doses of the pharmaceutical composition includes administration of the multi-specific binding protein at an amount selected from 5.2 × 10
-5 mg/kg, 1.6 × 10
-4 mg/kg, 5.2 × 10
-4 mg/kg, 1.6 × 10
-3 mg/kg, 5.2 × 10
-3 mg/kg, 1.6 × 10
-2 mg/kg, 5.2 × 10
-2 mg/kg, 1.6 × 10
-1 mg/kg, 0.52 mg/kg, 1.0 mg/kg, 1.6
mg/kg, 5.2 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg. In certain embodiments, the pharmaceutical composition is administered by intravenous infusion. In certain embodiments, the multi-specific binding protein is administered subcutaneously. [0026] In certain embodiments, the pharmaceutical composition is used as a monotherapy. [0027] In other embodiments, the method further includes administering to the subject an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, 200 mg of pembrolizumab is administered on Day 1 of the initial treatment cycle. In certain embodiments, if the subject receives one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles. [0028] In certain embodiments, a corticosteroid is administered to the subject on Day 1 of the initial four-week cycle. In certain embodiments, an antihistamine is administered to the subject on Day 1 of the initial four-week cycle. In certain embodiments, an antipyretic is administered to the subject on Day 1 of the initial four-week cycle. [0029] In another aspect, the present disclosure provides a pharmaceutical composition containing greater than 50 mg/mL of a multi-specific binding protein histidine, a sugar or sugar alcohol, and a polysorbate, at pH 5.5 to 6.5. The multi-specific binding protein incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0030] In some embodiments of any one of the preceding pharmaceutical compositions or methods using pharmaceutical compositions, the pharmaceutical composition contains greater than or equal to 60 mg/mL, greater than or equal to 70 mg/mL, greater than or equal to 80 mg/mL, greater than or equal to 90 mg/mL, greater than or equal to 100 mg/mL, greater than or equal to 125 mg/mL, greater than or equal to 150 mg/mL, greater than or equal to 175 mg/mL, or greater than or equal to 200 mg/mL of the multi-specific binding protein. In some embodiments, the pharmaceutical composition contains 60-250 mg/mL, 60-225 mg/mL, 60- 200 mg/mL, 60-175 mg/mL, 50-150 mg/mL, 60-150 mg/mL, 60-125 mg/mL, 60-100 mg/mL, 60-90 mg/mL, 60-80 mg/mL, 60-70 mg/mL, 70-250 mg/mL, 70-225 mg/mL, 70-200 mg/mL, 70-175 mg/mL, 70-150 mg/mL, 70-150 mg/mL, 70-125 mg/mL, 70-100 mg/mL, 70-90 mg/mL, 70-80 mg/mL, 80-250 mg/mL, 80-225 mg/mL, 80-200 mg/mL, 80-175 mg/mL, 80-
150 mg/mL, 80-150 mg/mL, 80-125 mg/mL, 80-100 mg/mL, 80-90 mg/mL, 90-250 mg/mL, 90-225 mg/mL, 90-200 mg/mL, 90-175 mg/mL, 90-150 mg/mL, 90-150 mg/mL, 90-125 mg/mL, 90-100 mg/mL, 100-250 mg/mL, 100-225 mg/mL, 100-200 mg/mL, 100-175 mg/mL, 100-150 mg/mL, 100-125 mg/mL, 125-250 mg/mL, 125-225 mg/mL, 125-200 mg/mL, 125-175 mg/mL, 125-150 mg/mL, 150-250 mg/mL, 150-225 mg/mL, 150-200 mg/mL, 150-175 mg/mL, 175-250 mg/mL, 175-225 mg/mL, 175-200 mg/mL, 200-250 mg/mL, or 200-225 mg/mL of the multi-specific binding protein. In some embodiments, the pharmaceutical composition contains 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, or 220 mg/mL of the multi- specific binding protein. [0031] In some embodiments, the pH of the pharmaceutical composition is 5.8 to 6.2. In some embodiments, the pH of the pharmaceutical composition is 5.95 to 6.05. In some embodiments, the concentration of histidine in the pharmaceutical composition is 10 to 25 mM. In some embodiments, the concentration of histidine in the pharmaceutical composition is about 20 mM. [0032] In some embodiments, the sugar or sugar alcohol is a disaccharide. In some embodiments, the disaccharide is sucrose. In some embodiments, the sugar or sugar alcohol is a sugar alcohol derived from a monosaccharide. In some embodiments, the sugar alcohol derived from a monosaccharide is sorbitol. In some embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical composition is 200 to 300 mM. In some embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM. [0033] In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical composition is 0.005% (w/v) to 0.05% (w/v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical composition is about 0.01% (w/v). In some embodiments, the concentration of NaCl, if any, is about 10 mM or lower in the pharmaceutical formulation. In some embodiments, the concentration of NaCl, if any, is about 1 mM or lower in the pharmaceutical formulation. [0034] In some embodiments, the first antigen-binding site includes a heavy chain variable domain with the complementarity-determining region 1 (CDR1), complementarity- determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences
represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively, and a light chain variable domain with the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In some embodiments, the heavy chain variable domain has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 169, respectively; and the light chain variable domain has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. [0035] In some embodiments, the heavy chain variable domain of the first antigen- binding site has an amino acid sequence at least 90% identical to SEQ ID NO:94, and the light chain variable domain has an amino acid sequence at least 90% identical to SEQ ID NO:98. In some embodiments, the heavy chain variable domain of the first antigen-binding site has the amino acid sequence of SEQ ID NO:94, and the light chain variable domain has the amino acid sequence of SEQ ID NO:98. In some embodiments, the first antigen-binding site that binds NKG2D is a Fab. [0036] In some embodiments, the second antigen-binding site includes a heavy chain variable domain with the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively, and a light chain variable domain with the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively. [0037] In some embodiments, the heavy chain variable domain of the second antigen- binding site has an amino acid sequence at least 90% identical to SEQ ID NO:195, and the light chain variable domain of the second antigen-binding site has an amino acid sequence at least 90% identical to SEQ ID NO:196. In some embodiments, the heavy chain variable domain of the second antigen-binding site has the amino acid sequence of SEQ ID NO:195, and the light chain variable domain of the second antigen-binding site has the amino acid sequence of SEQ ID NO:196. [0038] In some embodiments, the second antigen-binding site is an scFv. In some embodiments, the light chain variable domain of the second antigen-binding site is linked to the heavy chain variable domain of the second antigen-binding site via a flexible linker. In some embodiments, the flexible linker has the amino acid sequence of SEQ ID NO:143. [0039] In some embodiments, the light chain variable domain of the second antigen- binding site is positioned to the N-terminus of the heavy chain variable domain of the second
antigen-binding site. In some embodiments, the heavy chain variable domain of the second antigen-binding site forms a disulfide bridge with the light chain variable domain of the second antigen-binding site. In some embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, the amino acid positions numbered under Kabat. In some embodiments, the cysteine residues at position 44 of the heavy chain variable domain and at position 100 of the light chain variable domain are introduced by mutating the wild-type residues to cysteine residues at these positions. In some embodiments, the scFv has the amino acid sequence of SEQ ID NO:139. [0040] In some embodiments, the multi-specific binding protein includes an antibody Fc domain. In some embodiments, the antibody Fc domain includes a first antibody Fc polypeptide linked to the first antigen-binding site and a second antibody Fc polypeptide linked to the second antigen-binding site. In some embodiments, the first antibody Fc polypeptide is linked to the heavy chain portion of the first antigen-binding site. In some embodiments, the second antigen-binding site is linked to the second antibody Fc polypeptide via a hinge including Ala-Ser. In some embodiments, the first and second antibody Fc polypeptides each include a hinge and a CH2 domain of a human IgG1 antibody. In some embodiments, the first and second antibody Fc polypeptides each have an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody. In some embodiments, the first and second antibody Fc polypeptides incorporate different mutations promoting heterodimerization. In some embodiments, the first antibody Fc polypeptide is a human IgG1 Fc sequence having K360E and K409W substitutions. In some embodiments, the second antibody Fc polypeptide is a human IgG1 Fc sequence having Q347R, D399V, and F405T substitutions. [0041] In some embodiments, the multi-specific binding protein binds HER2 and includes a first polypeptide having the amino acid sequence of SEQ ID NO:141, a second polypeptide having the amino acid sequence of SEQ ID NO:140, and a third polypeptide having the amino acid sequence of SEQ ID NO:142. [0042] In another aspect, the present disclosure provides a method of treating a HER2- overexpressing gastric cancer in a subject in need thereof by administering to the subject a multi-specific binding protein. The multi-specific binding protein incorporates: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.
[0043] In certain embodiments, the gastric cancer is advanced gastric cancer or cancer of the gastro-esophageal junction. In certain embodiments, the subject previously received a first line of therapy. In certain embodiments, the first line of therapy included a platinum salt and a fluoropyridine in combination with trastuzumab or a biosimilar to trastuzumab. In certain embodiments, the subject progressed after the first line of therapy. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., determined by in situ hybridization). [0044] In certain embodiments, the method of treating gastric cancer further includes administering an anti-PD-1 antibody to the subject. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, nivolumab is administered to the subject at a dose of 120 to 600 mg. In certain embodiments, nivolumab is administered to the subject at a dose of 480 mg. In certain embodiments, nivolumab is administered once every four weeks. In certain embodiments, the multi-specific binding protein is administered in one or more four-week treatment cycles, and nivolumab is administered on Day 1 of the same treatment cycles. In certain embodiments, nivolumab is administered to the subject by intravenous infusion. [0045] In certain embodiments, the method of treating gastric cancer further includes administering a cytoskeletal-disrupting chemotherapeutic agent to the subject. In certain embodiments, the cytoskeletal-disrupting chemotherapeutic agent is nab-paclitaxel. In certain embodiments, nab-paclitaxel is administered to the subject at a dose of 50 to 300 mg/m
2. In certain embodiments, nab-paclitaxel is administered to the subject at a dose of 100 mg/m
2. In certain embodiments, nab-paclitaxel is administered three times every four weeks. In certain embodiments, the multi-specific binding protein is administered in one or more four-week treatment cycles, and nab-paclitaxel is administered on Day 1, Day 8, and Day 15 of the same treatment cycles. In certain embodiments, nab-paclitaxel is not administered on Day 22 of the treatment cycles. In certain embodiments, nab-paclitaxel is administered to the subject by intravenous infusion. [0046] In another aspect, the present disclosure provides a method of treating triple- negative breast cancer in a subject in need thereof by administering to the subject a multi- specific binding protein. The multi-specific binding protein incorporates: (a) a first antigen-
binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. [0047] In certain embodiments, the subject is not eligible for treatment in combination with an anti-PD-L1 therapy or the cancer in the subject is PD-L1 negative. In certain embodiments, the subject (a) is eligible for treatment with nab-paclitaxel after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy; (b) has a cancer that has no standard therapy or for which standard therapy has failed; or (c) has an advanced, optionally unresectable, recurrent, or metastatic triple- negative breast cancer. In certain embodiments, the triple-negative breast cancer has a PD- L1 score (CPS) less than 10 as measured by immunohistochemistry. In certain embodiments, the triple-negative breast cancer is metastatic or locally advanced. In certain embodiments, the subject has not previously received a chemotherapy or targeted systemic therapy. [0048] In certain embodiments of the method of treating gastric cancer or triple- negative breast cancer, the multi-specific binding protein is administered on Day 1, Day 8, and Day 15 of an initial treatment cycle. In certain embodiments, the multi-specific binding protein is not administered on Day 22 of the initial treatment cycle. In certain embodiments, after the initial treatment cycle, the multi-specific binding protein is administered on Day 1 and Day 15 of one or more subsequent four-week treatment cycles. In certain embodiments, the multi-specific binding protein is administered, in each dose, at an amount selected from the group consisting of 5.2 × 10
-5 mg/kg, 1.6 × 10
-4 mg/kg, 5.2 × 10
-4 mg/kg, 1.6 × 10
-3 mg/kg, 5.2 × 10
-3 mg/kg, 1.6 × 10
-2 mg/kg, 5.2 × 10
-2 mg/kg, 1.6 × 10
-1 mg/kg, 0.52 mg/kg, 1.0 mg/kg, 1.6 mg/kg, 5.2 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, and 50 mg/kg. In certain embodiments, the multi-specific binding protein is administered by intravenous infusion. In certain embodiments, the multi-specific binding protein is used as a monotherapy. [0049] In certain embodiments, the first antigen-binding site takes the format of a Fab having a heavy chain variable domain (VH) and a light chain variable domain (VL). The VH of the Fab has complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively. The VL of the Fab has CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In certain embodiments, the VH of the Fab has
CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 169, respectively. In certain embodiments, the VL of the Fab has CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In certain embodiments, the VH of the Fab has an amino acid sequence at least 90% identical to SEQ ID NO:94, and the VL of the Fab has an amino acid sequence at least 90% identical to SEQ ID NO:98. In certain embodiments, the VH of the Fab has the amino acid sequence of SEQ ID NO:94, and the VL of the Fab has the amino acid sequence of SEQ ID NO:98. [0050] In certain embodiments, the second antigen-binding site takes the format of a single chain variable fragment (scFv) having a VH and a VL. The VH of the scFv has CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively. The VL of the scFv has CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively. In certain embodiments, the VH of the scFv has an amino acid sequence at least 90% identical to SEQ ID NO:195, and the VL of the scFv has an amino acid sequence at least 90% identical to SEQ ID NO:196. In certain embodiments, the VH of the scFv has the amino acid sequence of SEQ ID NO:195, and the VL of the scFv has the amino acid sequence of SEQ ID NO:196. In certain embodiments, the VL of the scFv is linked to the VH of the scFv via a flexible linker, for example, a flexible linker having the amino acid sequence of SEQ ID NO:143. In certain embodiments, the VL of the scFv is positioned to the N-terminus of the VH of the scFv. In certain embodiments, the VH of the scFv forms a disulfide bridge with the VL of the scFv, for example, a disulfide bridge formed between C44 of the VH and C100 of the VL. In certain embodiments, the scFv has the amino acid sequence of SEQ ID NO:139. [0051] In certain embodiments, the antibody Fc domain includes a first antibody Fc sequence linked to the Fab and a second antibody Fc sequence linked to the scFv. In certain embodiments, the first antibody Fc sequence is linked to the heavy chain portion of the Fab. In certain embodiments, the scFv is linked to the second antibody Fc sequence via a hinge having the amino acid sequence Ala-Ser. In certain embodiments, the first and second antibody Fc sequences each has a hinge and a CH2 domain of a human IgG1 antibody. In certain embodiments, the first and second antibody Fc sequences each has an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody.
In certain embodiments, the first and second antibody Fc sequences have different mutations that promote heterodimerization. For example, in certain embodiments, the first antibody Fc sequence is a human IgG1 Fc sequence incorporating K360E and K409W substitutions, and/or the second antibody Fc sequence is a human IgG1 Fc sequence incorporating Q347R, D399V, and F405T substitutions. [0052] In certain embodiments, the multi-specific binding protein includes (a) a first polypeptide having the amino acid sequence of SEQ ID NO:141, (b) a second polypeptide having the amino acid sequence of SEQ ID NO:140, and (c) a third polypeptide having the amino acid sequence of SEQ ID NO:142. [0053] In certain embodiments, the multi-specific binding protein, prior to the administration, has been formulated in a pharmaceutical composition at a concentration of 10 mg/mL to 50 mg/mL. In certain embodiments, the pharmaceutical composition contains 10 mg/mL to 25 mg/mL of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition contains about 15 mg/mL of the multi-specific binding protein. [0054] In certain embodiments, the pharmaceutical composition has a pH in the range of 5.5 to 6.5, for example, a pH of about 6.0. In certain embodiments, the pharmaceutical composition contains 5 to 50 mM histidine. In certain embodiments, the pharmaceutical composition contains 10 to 25 mM histidine. In certain embodiments, the pharmaceutical composition contains about 20 mM histidine. [0055] In certain embodiments, the pharmaceutical composition contains 50 to 300 mM sugar or sugar alcohol (e.g., sucrose). In certain embodiments, the pharmaceutical composition contains 150 to 300 mM of the sugar or sugar alcohol. In certain embodiments, the pharmaceutical composition contains about 250 mM of the sugar or sugar alcohol. [0056] In certain embodiments, the pharmaceutical composition contains 0.005% to 0.05% mM (w/v) polysorbate (e.g., polysorbate-80). In certain embodiments, the pharmaceutical composition contains 0.005% to 0.02% mM (w/v) of the polysorbate. In certain embodiments, the pharmaceutical composition contains about 0.01% (w/v) of the polysorbate. [0057] In certain embodiments, the pharmaceutical composition contains (i) 10 mg/mL to 50 mg/mL of the multi-specific binding protein, (ii) 5 mM to 50 mM histidine, (iii) 50 mM
to 300 mM sucrose, and (iv) 0.005% to 0.05% (w/v) polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition contains (i) 10 mg/mL to 25 mg/mL of the multi-specific binding protein, (ii) 10 mM to 25 mM histidine, (iii) 150 mM to 300 mM sucrose, and (iv) 0.005% to 0.02% (w/v) polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition contains (i) about 15 mg/mL of the multi- specific binding protein, (ii) about 20 mM histidine, (iii) about 250 mM sucrose, and (iv) about 0.01% (w/v) polysorbate 80, at about pH 6.0. [0058] Other embodiments and details of the disclosure are presented herein below. BRIEF DESCRIPTION OF THE DRAWINGS [0059] FIG. 1 is a representation of a heterodimeric, multi-specific antibody, e.g., a trispecific binding protein (TriNKET). Each arm can represent either an NKG2D binding domain or a tumor-associated antigen binding domain. In some embodiments, the NKG2D binding domain and the tumor-associated antigen binding domain can share a common light chain. [0060] FIGs. 2A-2E illustrate five exemplary formats of a multi-specific binding protein, e.g., a trispecific binding protein (TriNKET). As shown in FIG. 2A, either the NKG2D-binding domain or the tumor-associated antigen binding domain can take the scFv format (left arm). An antibody that contains an NKG2D targeting scFv, a tumor-associated antigen targeting Fab fragment, and a heterodimerized antibody constant region is referred herein as the F3-TriNKET. An antibody that contains a tumor-associated antigen targeting scFv, an NKG2D targeting Fab fragment, and a heterodimerized antibody constant region/domain that binds CD16 is referred herein as the F3’-TriNKET (FIG. 2E). As shown in FIG. 2B, both the NKG2D binding domain and a tumor-associated antigen binding domain can take the scFv format. FIGs. 2C to 2D are illustrations of an antibody with three antigen-binding sites, including two antigen-binding sites that bind a tumor-associated antigen, and the NKG2D-binding site fused to the heterodimerized antibody constant region. These antibody formats are referred herein as F4-TriNKET. FIG. 2C illustrates that the two tumor-associated antigen binding sites are in the Fab fragment format, and the NKG2D binding site in the scFv format. FIG. 2D illustrates that the tumor-associated antigen binding sites are in the scFv format, and the NKG2D binding site is in the scFv format. FIG. 2E represents a trispecific antibody (TriNKET) that contains a tumor-associated antigen targeting scFv, an NKG2D targeting Fab fragment, and a heterodimerized antibody constant region/domain (“CD domain”) that binds CD16. The antibody format is referred herein as
F3’-TriNKET. In certain exemplary multi-specific binding proteins, heterodimerization mutations on the antibody constant region include K360E and K409W on one constant domain; and Q347R, D399V and F405T on the opposite constant domain (shown as a triangular lock-and-key shape in the CD domains). The bold bar between the heavy and the light chain variable domains of the Fab fragments represents a disulfide bond. [0061] FIG. 3 is a representation of a TriNKET in the Triomab form, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light and one heavy chain, that originate from two parental antibodies. Triomab form may be a heterodimeric construct containing 1/2 of rat antibody and 1/2 of mouse antibody. [0062] FIG. 4 is a representation of a TriNKET in the KiH Common Light Chain form, which involves the knobs-into-holes (KIHs) technology. KiH is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc domain stabilized by heterodimerization mutations. TriNKET in the KiH format may be a heterodimeric construct with 2 Fab fragments binding to target 1 and target 2, containing two different heavy chains and a common light chain that pairs with both heavy chains. [0063] FIG. 5 is a representation of a TriNKET in the dual-variable domain immunoglobulin (DVD-Ig™) form, which combines the target-binding domains of two monoclonal antibodies via flexible naturally occurring linkers, and yields a tetravalent IgG- like molecule. DVD-Ig™ is a homodimeric construct where variable domain targeting antigen 2 is fused to the N-terminus of a variable domain of a Fab fragment targeting antigen 1. DVD-Ig™ form contains a normal Fc domain. [0064] FIG. 6 is a representation of a TriNKET in the Orthogonal Fab fragment interface (Ortho-Fab) form, which is a heterodimeric construct that contains 2 Fab fragments binding to target 1 and target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by orthogonal interface. Heterodimerization is ensured by mutations in the Fc domain. [0065] FIG. 7 is a representation of a TriNKET in the 2-in-1 Ig format. [0066] FIG. 8 is a representation of a TriNKET in the ES form, which is a heterodimeric construct containing two different Fab fragments binding to target 1 and target 2 fused to the Fc domain. Heterodimerization is ensured by electrostatic steering mutations in the Fc domain. [0067] FIG. 9 is a representation of a TriNKET in the Fab Arm Exchange form: antibodies that exchange Fab fragment arms by swapping a heavy chain and attached light
chain (half-molecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies. Fab Arm Exchange form (cFae) is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc domain stabilized by heterodimerization mutations. [0068] FIG. 10 is a representation of a TriNKET in the SEED Body form, which is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc domain stabilized by heterodimerization mutations. [0069] FIG. 11 is a representation of a TriNKET in the LuZ-Y form, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs binding to target 1 and 2, fused to an Fc domain. Heterodimerization is ensured through leucine zipper motifs fused to the C-terminus of the Fc domain. [0070] FIG. 12 is a representation of a TriNKET in the Cov-X-Body form. [0071] FIGs. 13A-13B are representations of TriNKETs in the ĸλ-Body forms, which are heterodimeric constructs with two different Fab fragments fused to a Fc domain stabilized by heterodimerization mutations: one Fab fragment targeting antigen 1 contains kappa LC, and the second Fab fragment targeting antigen 2 contains lambda LC. FIG. 13A is an exemplary representation of one form of a ĸλ-Body; FIG. 13B is an exemplary representation of another ĸλ-Body. [0072] FIG. 14 is a representation of an Oasc-Fab heterodimeric construct that includes a Fab fragment binding to target 1 and an scFab binding to target 2, both of which are fused to the Fc domain. Heterodimerization is ensured by mutations in the Fc domain. [0073] FIG. 15 is a representation of a DuetMab, which is a heterodimeric construct containing two different Fab fragments binding to antigens 1 and 2, and an Fc domain that is stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct light chain and heavy chain pairing. [0074] FIG. 16 is a representation of a CrossmAb, which is a heterodimeric construct with two different Fab fragments binding to targets 1 and 2, and an Fc domain stabilized by heterodimerization mutations. CL and CH1 domains, and VH and VL domains are switched, e.g., CH1 is fused in-line with VL, and CL is fused in-line with VH. [0075] FIG. 17 is a representation of a Fit-Ig, which is a homodimeric construct where a Fab fragment binding to antigen 2 is fused to the N-terminus of HC of a Fab fragment that binds to antigen 1. The construct contains a wild-type Fc domain.
[0076] FIG. 18A is an interaction plot for average size as measured by Dynamic Light Scattering (DLS) after a 3-week incubation at 50 °C. FIG. 18B is an interaction plot for average size as measured by DLS after a 3-week incubation at 2-8 °C. [0077] FIG. 19A is an interaction plot for monomer size as measured by DLS after a 3- week incubation at 50 °C. FIG. 19B is an interaction plot for monomer size as measured by DLS after a 3-week incubation at 2-8 °C. [0078] FIG. 20A is an interaction plot for % main species determined by Size Exclusion Chromatography (SEC) for a 3-week incubation at 50 °C. FIG. 20B is an interaction plot for % main species determined by SEC for a 3-week incubation at 2-8 °C. [0079] FIG. 21A is an interaction plot for percent High Molecular Weight (%HMW) species determined by SEC for a 3-week incubation at 50 °C. FIG. 21B is an interaction plot for %HMW species determined by SEC for a 3-week incubation at 2-8 °C. [0080] FIG. 22A is an interaction plot for percent Low Molecular Weight (%LMW) species determined by SEC for a 3-week incubation at 50 °C. FIG. 22B is an interaction plot for %LMW species determined by SEC for a 3-week incubation at 2-8 °C at pH 6.0. [0081] FIG. 23A is an interaction plot for % acidic species determined by Imaged Capillary Isoelectric Focusing (icIEF) for a 3-week incubation at 50 °C. FIG. 23B is an interaction plot for % basic species for sucrose only as determined by icIEF for a 3-week incubation 4–8 ˚C. [0082] FIG. 24A is an interaction plot for % main species determined by icIEF for a 3- week incubation at 50 °C. FIGs. 24B-24D are interaction plots for % main species in the sucrose only formulation, determined by icIEF, for a 3-week incubation at 4 °C at pH 5.5 (FIG. 24B), pH 6.0 (FIG. 24C), and pH 6.5 (FIG. 24D). [0083] FIG. 25A is an interaction plot for % basic species determined by icIEF for a 3- week incubation at 50 °C. FIG. 25B is an interaction plot for % basic species for sucrose only as determined by icIEF for a 3-week incubation 2–8 ˚C. [0084] FIG. 26A is an interaction plot for % purity determined by Capillary Electrophoresis (CE) for a 3-week incubation at 50 °C. FIG. 26B is an interaction plot for % impurities determined by CE for a 3-week incubation at 50 °C. [0085] FIG. 27A is an interaction plot for %main species determined by Capillary Electrophoresis (Non-Reduced) (CE (NR)) for a 3-week incubation at 50 °C at pH 6.0. FIG.
27B is an interaction plot for %main species determined by CE (NR) for a 3-week incubation at 2–8 °C at pH 6.0. [0086] FIG. 28A is an interaction plot for %HMW species determined by CE (NR) for a 3-week incubation at 50 °C. FIG. 28B shows the interaction plot for %HMW species determined by CE (NR) for a 3-week incubation at 2–8 °C. [0087] FIG. 29A is an interaction plot for %LMW species for sucrose only as determined by CE (NR) for a 3-week incubation at 50 °C. FIG. 29B is an interaction plot for %LMW species for sucrose only as determined by CE (NR) for a 3-week incubation at 2-8 °C. [0088] FIG. 30 is a graph of concentration curves of A49-F3’-TriNKET-Trastuzumab over time in formulations stored at 2-8°C or 25°C. [0089] FIG. 31A is an image of vials containing formulations of A49-F3’-TriNKET- Trastuzumab in varying concentrations of polysorbate-80. FIG. 31B is a graph of % Monomer species of A49-F3’-TriNKET-Trastuzumab in formulations stored at varying temperatures at specific time points as measured by size-exclusion high-performance liquid chromatography (SE-HPLC). [0090] FIGs. 32A-32B is a schematic diagram of a clinical trial design. FIG. 32A describes a trial design for a dose escalation phase. FIG. 32B describes a trial design for an efficacy expansion cohorts phase. Abbreviations used in the figures include: DL=dose level; Combo PD-1=combination therapy with pembrolizumab; PK=pharmacokinetics; PD=pharmacodynamics; HER2 HIGH=high expression of HER2 of 3+, per immunohistochemistry; MBC HER22+/1+=metastatic breast cancer with medium/low expression of HER2 of 2+/1+, per immunohistochemistry; UBC 2L/3L=urothelial bladder cancer 2nd line-/3rd line treatment. [0091] FIGs. 33A-33C are line graphs showing the cytotoxicity of A49-F3’-TriNKET- Trastuzumab and trastuzumab on SKBR-3 (FIG. 33A), H661 (FIG. 33B), and 786-O (FIG. 33C) cancer cells in the presence of NK cells. [0092] FIGs. 34A-34B are line graphs showing the cytotoxicity of A49-F3’-TriNKET- Trastuzumab and trastuzumab on ZR-75-1 (FIG. 34A) and MCF-7 (FIG. 34B) cancer cells in the presence of NK cells.
[0093] FIGs. 35A-35C are line graphs showing the cytotoxicity of A49-F3’-TriNKET- Trastuzumab and trastuzumab on BT-20 (FIG. 35A), Hs578T (FIG. 35B) and DU4475 (FIG. 35C) cancer cells in the presence of KHYG-1-CD16V cells. DETAILED DESCRIPTION Definitions [0094] To facilitate an understanding of the present invention, a number of terms and phrases are defined below. [0095] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. [0096] As used herein, the terms “Fab” and “scFv” refer to two different forms of protein fragments that each include an antigen-binding site. The term “antigen-binding site” refers to the part of the immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N- terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains, which are also called “VH” and “VL,” respectively. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FR.” Thus the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen- binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a “single domain antibody.” Antigen-binding sites can exist in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen- binding surface such as a Fab, or in a recombinant polypeptide such as an scFv, using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide. All the amino acid positions in heavy or light chain variable regions disclosed herein are numbered according to Kabat numbering, unless otherwise indicated.
[0097] The CDRs of an antigen-binding site can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). The CDRs determined under these definitions typically include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609- 6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, heavy chain CDRs and light chain CDRs of an antibody are defined using different conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to MacCallum (supra), and the light CDRs are defined according to Kabat (supra). CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 denote the light chain CDRs. [0098] The term “tumor-associated antigen” or “TAA,” as used herein, means any antigen including but not limited to a protein, glycoprotein, ganglioside, carbohydrate, or lipid that is associated with cancer. In certain embodiments, a tumor-associated antigen is expressed on the surface of a cell. For example, a tumor-associated antigen can be expressed on malignant cells or in the tumor microenvironment, such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. [0099] The term “HER2-overexpressing,” as used herein to characterize a cancer, means HER2-positive cancer according to the ASCO/CAP HER2 testing guideline (Wolff et al., (2007) J. Clin. Oncol. 25(1):118-45) and the 2018 update (Wolff et al., (2018) J. Clin. Oncol. 36(20):2105-22). A HER2-overexpressing cancer can have a HER2 expression level scored as 3+ by immunohistochemistry, or a HER2 expression level scored as 2+ by immunohistochemistry further supported by detection of ERBB2 gene amplification (e.g., determined by in situ hybridization (ISH), chromogenic in situ hybridization (CISH), quantitative PCR, or DNA sequencing). [0100] As used herein, the term “pharmaceutical formulation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
[0101] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, primates, canines, felines, and the like), and more preferably include humans. [0102] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. [0103] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present invention) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. [0104] The term “about” refers to any minimal alteration in the concentration or amount of an agent that does not change the efficacy of the agent in preparation of a formulation and in treatment of a disease or disorder. In certain embodiments, the term “about” may include ±5%, ±10%, or ±15% of a specified numerical value or data point. [0105] Ranges can be expressed in this disclosure as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed in this
disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. [0106] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited steps. [0107] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls. Multi-Specific Binding Proteins [0108] The present disclosure provides pharmaceutical compositions or pharmaceutical formulations that contain a multi-specific binding protein having an antigen-binding site that binds a tumor-associated antigen such as HER2, an antigen-binding site that binds NKG2D, and an antibody Fc domain. Also provided are uses of the multi-specific binding proteins and pharmaceutical formulations in treating cancer, such as a locally advanced or metastatic solid tumor. The multi-specific binding proteins are capable of binding a target (e.g., HER2) on a cancer cell and NKG2D and CD16 on natural killer cells. Such binding brings the cancer cell into proximity with the natural killer cell, which facilitates direct and indirect destruction of the cancer cell by the natural killer cells. [0109] The first component of the multi-specific binding proteins binds to NKG2D receptor-expressing cells, which can include but are not limited to NK cells, NKT cells, γδ T
cells and CD8
+ αβ T cells. Upon NKG2D binding, the multi-specific binding proteins may block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NK cells. The second component of the multi-specific binding proteins binds to HER2- expressing cells, which can include but are limited to breast, ovarian, esophageal, bladder and gastric cancer, salivary duct carcinoma, adenocarcinoma of the lung and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. The third component of the multi-specific binding proteins is an antibody Fc domain, which binds to cells expressing CD16 such as NK cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. [0110] The multi-specific binding proteins described herein can take various formats. For example, one format involves a heterodimeric, multi-specific antibody including a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain includes a first Fc (hinge-CH2- CH3) domain fused via either a linker or an antibody hinge to an Fab fragment composed of a heavy chain portion including a heavy chain variable domain and a heavy chain CH1 domain, and a light chain portion including a light chain variable domain and a light chain constant domain (CL), and the heavy chain and light chain portions of the Fab fragment pair and bind NKG2D. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of a heavy chain variable domain and light chain variable domain which pair and bind the tumor-associate antigen. [0111] In some embodiments, the single-chain variable fragment (scFv) described above is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge has the amino acid sequence Ala-Ser. In some other embodiments, the hinge has the amino acid sequences Ala-Ser and Thr-Lys-Gly. The hinge sequence can provide flexibility of binding to the target antigen, and balance between flexibility and optimal geometry. [0112] In some embodiments, the single-chain variable fragment (scFv) described above includes a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance stability of the scFv. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions numbered under Kabat. For example, in some embodiments, the disulfide bridge is formed between a cysteine residue
(naturally present or introduced by mutation) at position 44 (C44) of the VH of the scFv and a cysteine residue (naturally present or introduced by mutation) at position 100 (C100) of the VL of the scFv, numbered under the Kabat numbering scheme. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker can be used, for example, the (G4S)
4 linker (SEQ ID NO:143). In some embodiments of the scFv, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain. In some embodiments of the scFv, the heavy chain variable domain is positioned at the C terminus of the light chain variable domain. [0113] The multi-specific binding proteins described herein can further include one or more additional antigen-binding sites. The additional antigen-binding site(s) may be fused to the C-terminus of the constant region CH2 domain or to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the additional antigen-binding site(s) takes the form of a single-chain variable region (scFv) that is optionally disulfide-stabilized, resulting in a tetravalent or trivalent multi-specific binding protein. For example, a multi-specific binding protein includes an NKG2D-binding site, a TAA-binding site, a third antigen-binding site that binds a TAA, and an antibody constant region or a portion thereof sufficient to bind CD16, or a fourth antigen-binding site that binds CD16. Any one of these antigen-binding sites can either take the form of an Fab or an scFv, such as the scFv described above. In some embodiments, the third antigen-binding site binds a different TAA. In some embodiments, the third antigen-binding site binds to the same TAA, and the exemplary formats are shown in FIGs. 2C and 2D. Accordingly, the multi- specific binding proteins can provide bivalent engagement of the TAA. In certain embodiments, bivalent engagement of the TAA by the multi-specific proteins can stabilize the TAA on the cancer cell surface, and enhance cytotoxicity of NK cells towards the cancer cells. Bivalent engagement of the TAA by the multi-specific proteins can confer stronger binding of the multi-specific proteins to the cancer cells, thereby facilitating a stronger cytotoxic response of NK cells towards the cancer cells, especially towards cancer cells expressing a low level of the TAA. [0114] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is primarily focused on amino acid residues Asp 265 – Glu 269, Asn 297 – Thr 299, Ala 327 – Ile 332, Leu 234 – Ser 239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., Nature, 406 (6793):267-273). Based on the known domains,
mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using phage-displayed libraries or yeast surface-displayed cDNA libraries, or can be designed based on the known three-dimensional structure of the interaction. [0115] In some embodiments, the antibody constant domain includes a CH2 domain and a CH3 domain of an IgG antibody, for example, a human IgG1 antibody. In some embodiments, mutations are introduced in the antibody constant domain to enable heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from the constant domain of a human IgG1, the antibody constant domain can have an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody that differs at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. All the amino acid positions in an Fc domain or hinge region disclosed herein are numbered according to EU numbering. [0116] In some embodiments, the antibody constant domain can have an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody that differs by one or more substitutions selected from Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E. [0117] Individual components of the multi-specific binding proteins are described in more detail below. NKG2D-Binding Site [0118] Upon binding to the NKG2D receptor and CD16 receptor on natural killer cells and a TAA on cancer cells, the multi-specific binding proteins can engage more than one kind of NK-activating receptor, and may block the binding of natural ligands to NKG2D. In certain embodiments, the multi-specific binding proteins can agonize NK cells in humans. In some embodiments, the multi-specific binding proteins can agonize NK cells in humans and in other species such as rodents and cynomolgus monkeys.
[0119] Table 1 lists peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to NKG2D. In some embodiments, the heavy chain variable domain and the light chain variable domain are arranged in Fab format. These NKG2D binding domains can vary in their binding affinity to NKG2D, nevertheless, they all activate human NK cells. Unless indicated otherwise, the CDR sequences provided in Table 1 are determined under Kabat. Table 1. Exemplary NKG2D-Binding Sites
![Figure imgf000029_0001](https://patentimages.storage.googleapis.com/9e/b3/9d/631be956c50fbd/imgf000029_0001.png)
![Figure imgf000030_0001](https://patentimages.storage.googleapis.com/00/f8/84/fea5b24e3237cf/imgf000030_0001.png)
![Figure imgf000031_0001](https://patentimages.storage.googleapis.com/f9/27/1d/01c6a41834dc59/imgf000031_0001.png)
![Figure imgf000032_0001](https://patentimages.storage.googleapis.com/0a/51/8d/e594ae7eed26a8/imgf000032_0001.png)
![Figure imgf000033_0001](https://patentimages.storage.googleapis.com/01/d7/87/83b21864c6f87c/imgf000033_0001.png)
![Figure imgf000034_0001](https://patentimages.storage.googleapis.com/bf/28/84/260a731d1e4c5b/imgf000034_0001.png)
![Figure imgf000035_0001](https://patentimages.storage.googleapis.com/4c/ce/1e/9608e3d1d9a10a/imgf000035_0001.png)
![Figure imgf000036_0001](https://patentimages.storage.googleapis.com/6a/ca/df/402c1db3c1a87b/imgf000036_0001.png)
![Figure imgf000037_0001](https://patentimages.storage.googleapis.com/99/d3/9b/87fd99e7f1d7ab/imgf000037_0001.png)
![Figure imgf000038_0001](https://patentimages.storage.googleapis.com/bf/c0/b0/e9fc379c053c40/imgf000038_0001.png)
![Figure imgf000039_0001](https://patentimages.storage.googleapis.com/d5/49/54/633e778c0e8b59/imgf000039_0001.png)
![Figure imgf000040_0001](https://patentimages.storage.googleapis.com/87/d1/16/7dee719e25ab3b/imgf000040_0001.png)
[0188] Specific TriNKETs and their polypeptide chains are described in more detail below. In the amino acid sequences, (G
4S)
4 (SEQ ID NO:143) and Ala-Ser linkers are bold- underlined; Cys residues in the scFv that form disulfide bridges are bold-italic-underlined; Fc heterodimerization mutations are bold-underlined; and CDR sequences under Kabat are underlined.
[0189] For example, a TriNKET of the present disclosure is A49-F3’-TriNKET- Trastuzumab. A49-F3’-TriNKET-Trastuzumab includes a single-chain variable fragment (scFv) (SEQ ID NO:139) derived from trastuzumab that binds HER2, linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and an NKG2D-binding Fab fragment derived from A49 linked to a second antibody Fc polypeptide. The Fab fragment includes a heavy chain portion having a heavy chain variable domain (SEQ ID NO:94) and a CH1 domain, and a light chain portion having a light chain variable domain (SEQ ID NO:98) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A49-F3’- TriNKET-Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:140, SEQ ID NO:141, and SEQ ID NO:142. [0190] SEQ ID NO:140 represents the full sequence of the HER2-binding scFv linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv-Fc). The first antibody Fc polypeptide includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:141 as described below. The scFv (SEQ ID NO:139) includes a heavy chain variable domain of trastuzumab connected to the N-terminus of a light chain variable domain of trastuzumab via a (G
4S)
4 linker (SEQ ID NO:143), the scFv represented as VL-(G
4S)
4-VH (“(G
4S)
4” is represented by SEQ ID NO:143). The heavy and the light variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH, as a result of Q100C and G44C substitutions in the VL and VH, respectively. Trastuzumab scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:139) Trastuzumab scFv-Fc (RVT) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS
AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:140) [0191] SEQ ID NO:141 represents the heavy chain portion of the Fab fragment, which includes a heavy chain variable domain (SEQ ID NO:94) of an NKG2D-binding site and a CH1 domain, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:141 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc polypeptide in SEQ ID NO:140. In SEQ ID NO:141, the antibody Fc polypeptide also includes K360E and K409W substitutions for heterodimerization with the Fc in SEQ ID NO:140. A49 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSS (SEQ ID NO:94) A49 VH-CH1-Fc (EW) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:141) [0192] SEQ ID NO:142 represents the light chain portion of the Fab fragment including a light chain variable domain (SEQ ID NO:98) of an NKG2D-binding site and a light chain constant domain. A49 VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK (SEQ ID NO:98) A49 VL-LC DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK RTVAAPSPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:142) [0193] Another TriNKET of the present disclosure is A49MI-F3’-TriNKET- Trastuzumab. A49MI-F3’-TriNKET-Trastuzumab includes the same HER2-binding scFv (SEQ ID NO:139) as in A49-F3’-TriNKET-Trastuzumab linked via a hinge including Ala- Ser to an antibody Fc polypeptide; and an NKG2D-binding Fab fragment derived from A49MI linked to a second antibody Fc polypeptide. The Fab fragment includes a heavy chain portion including a heavy chain variable domain (SEQ ID NO:144) and a CH1 domain, and a light chain portion including a light chain variable domain (SEQ ID NO:98) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A49MI-F3’- TriNKET-Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:140 (as in A49-F3’-TriNKET-Trastuzumab), SEQ ID NO:145, and SEQ ID NO:142 (as in A49- F3’-TriNKET-Trastuzumab). [0194] SEQ ID NO:145 represents a heavy chain portion of the Fab fragment, which includes a heavy chain variable domain (SEQ ID NO:144) of an NKG2D-binding site and a CH1 domain, connected to the first antibody Fc polypeptide. In SEQ ID NO:144, a methionine in the CDR3 of SEQ ID NO:94 has been substituted by isoleucine (M Æ I substitution; shown within a third bracket [] in SEQ ID NO:144 and SEQ ID NO:145). The antibody Fc polypeptide in SEQ ID NO:145 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution in the antibody Fc polypeptide in SEQ ID NO:140. In SEQ ID NO:145, the antibody Fc polypeptide also includes K360E and K409W substitutions. A49MI VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAP[I]GAAAGWFDPW GQGTLVTVSS (SEQ ID NO:144) A49MI VH-CH1-Fc (EW) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAP[I]GAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:145) [0195] Another TriNKET of the present disclosure is A49-F3’-KiH-TriNKET- Trastuzumab. KiH refers to the knobs-into-holes (KiH) Fc technology, which involves engineering of the CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. The concept behind the KiH Fc technology was to introduce a “knob” in one CH3 domain (CH3A) by substitution of a small residue with a bulky one (e.g., T366W
CH3A in EU numbering). To accommodate the “knob,” a complementary “hole” surface was created on the other CH3 domain (CH3B) by replacing the closest neighboring residues to the knob with smaller ones (e.g., T366S/L368A/Y407V
CH3B). The “hole” mutation was optimized by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(1):26–35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9):1947–57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc domain variant with improved affinity for FcγRs. Mol. Immunol. (2014) 58(1):132–8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the inter-CH3 domain core interface, whereas the knob–knob and the hole–hole interfaces do not favor homodimerization owing to steric hindrance and disruption of the favorable interactions, respectively. [0196] A49-F3’-KiH-TriNKET-Trastuzumab includes the same HER2-binding scFv (SEQ ID NO:139) as in A49-F3’-TriNKET-Trastuzumab linked via a hinge including Ala- Ser to a first antibody Fc polypeptide incorporating the “hole” substitutions of T366S, L368A, and Y407V; and the same NKG2D-binding Fab fragment as in A49-F3’-TriNKET- Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide incorporating the “knob” substitution of T366W. A49-F3’-KiH-TriNKET-Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:146, SEQ ID NO:147, and SEQ ID NO:142 (as in A49-F3’-TriNKET-Trastuzumab). [0197] SEQ ID NO:146 represents the full sequence of the HER2-binding scFv (SEQ ID NO:139) linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv-
Fc). The first antibody Fc polypeptide includes T366S, L368A, and Y407V substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:147 as described below. Trastuzumab scFv-Fc (KiH) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:146) [0198] SEQ ID NO:147 represents the heavy chain portion of a Fab fragment having a heavy chain variable domain (SEQ ID NO:94) of an NKG2D-binding site derived from A49 and a CH1 domain, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:147 includes an S354C substitution, which forms a disulfide bond with a Y349C substitution in the CH3 domain of the first antibody Fc polypeptide. In SEQ ID NO:147, the antibody Fc polypeptide also includes a T366W substitution. A49 VH-CH1-Fc (KiH) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:147) [0199] Another TriNKET of the present disclosure is A49MI-F3’-KiH-TriNKET- Trastuzumab. A49MI-F3’-KiH-TriNKET-Trastuzumab includes the same HER2-binding scFv (SEQ ID NO:139) as in A49-F3’-TriNKET-Trastuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide incorporating the “hole” substitutions of T366S, L368A, and Y407V; and the same NKG2D-binding Fab fragment as in A49MI-F3’- TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc
polypeptide incorporating the “knob” substitution of T366W. A49MI-F3’-KiH-TriNKET- Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:146 (as in A49-F3’-KiH-TriNKET-Trastuzumab), SEQ ID NO:194, and SEQ ID NO:142 (as in A49- F3’-TriNKET-Trastuzumab). [0200] SEQ ID NO:194 represents the heavy chain portion of a Fab fragment having a heavy chain variable domain (SEQ ID NO:144) of an NKG2D-binding site derived from A49MI and a CH1 domain, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:194 includes an S354C substitution, which forms a disulfide bond with a Y349C substitution in the CH3 domain of the first antibody Fc polypeptide. In SEQ ID NO:194, the antibody Fc polypeptide also includes a T366W substitution. A49MI VH-CH1-Fc (KiH) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:194) [0201] Another exemplary TriNKET of the present disclosure is A44-F3’-TriNKET- Trastuzumab. A44-F3’-TriNKET-Trastuzumab includes the same HER2-binding scFv (SEQ ID NO:139) as in A49-F3’-TriNKET-Trastuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and an NKG2D-binding Fab fragment derived from A44 linked to a second antibody Fc polypeptide. The Fab fragment includes a heavy chain portion having a heavy chain variable domain (SEQ ID NO:86) and a CH1 domain, and a light chain portion having a light chain variable domain (SEQ ID NO:90) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A44-F3’-TriNKET- Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:140 (as in A49-F3’-TriNKET-Trastuzumab), SEQ ID NO:155, and SEQ ID NO:149. [0202] SEQ ID NO:155 represents a heavy chain variable domain (SEQ ID NO:86) of an NKG2D-binding site derived from A44, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:155 includes a Y349C substitution in the CH3
domain, which forms a disulfide bond with an S354C substitution on the first antibody Fc polypeptide. In SEQ ID NO:155, the antibody Fc polypeptide also includes K360E and K409W substitutions. A44 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYW GQGTLVTVSS (SEQ ID NO:86) A44 VH-CH1-Fc (EW) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:155) [0203] SEQ ID NO:149 represents the light chain portion of the Fab fragment including a light chain variable domain (SEQ ID NO:90) of an NKG2D-binding site and a light chain constant domain. A44 VL DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK (SEQ ID NO:90) A44 VL-CL DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:149) [0204] Another exemplary TriNKET of the present disclosure is A44-F3’-KiH- TriNKET-Trastuzumab. A44-F3’-KiH-TriNKET-Trastuzumab includes the same HER2- binding scFv (SEQ ID NO:139) as in A49-F3’-TriNKET-Trastuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide incorporating the “hole” substitutions of T366S, L368A, and Y407V; and the same NKG2D-binding Fab fragment as in A44-F3’- TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide incorporating the “knob” substitution of T366W. A44-F3’-KiH-TriNKET-
Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:146 (as in A49-F3’-KiH-TriNKET-Trastuzumab), SEQ ID NO:148, and SEQ ID NO:149 (as in A44- F3’-TriNKET-Trastuzumab). [0205] SEQ ID NO:148 represents a heavy chain variable domain (SEQ ID NO:86) of an NKG2D-binding site derived from A44, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:148 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the first antibody Fc polypeptide. In SEQ ID NO:148, the antibody Fc polypeptide also includes a T366W substitution. A44 VH-CH1-Fc (KiH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:148) [0206] Another TriNKET of the present disclosure is A49-F3’-TriNKET-Pertuzumab. A49-F3’-TriNKET-Pertuzumab includes an scFv (SEQ ID NO:189) derived from pertuzumab that binds HER2, linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49-F3’-TriNKET- Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49-F3’- TriNKET-Pertuzumab includes three polypeptides, having the sequences of SEQ ID NO:190, SEQ ID NO:141 (as in A49-F3’-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49- F3’-TriNKET-Trastuzumab). [0207] SEQ ID NO:190 represents the full sequence of the HER2-binding scFv linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv-Fc). The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:141 as described above. The scFv (SEQ ID NO:189) includes a heavy chain variable domain of pertuzumab connected to the N-terminus of a light chain
variable domain of pertuzumab via a (G
4S)
4 linker (SEQ ID NO:143), the scFv represented as VL-(G
4S)
4-VH (“(G
4S)
4” is represented by SEQ ID NO:143). The heavy and the light variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH, as a result of Q100C and G44C substitutions in the VL and VH, respectively. Pertuzumab scFv DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKCLEWVADVNPNS GGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQ GTLVTVSSA (SEQ ID NO:189) Pertuzumab scFv-Fc DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKCLEWVADVNPNS GGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQ GTLVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:190) [0208] Another exemplary TriNKET of the present disclosure is A49MI-F3’-TriNKET- Pertuzumab. A49MI-F3’-TriNKET-Pertuzumab includes the same HER2-binding scFv (SEQ ID NO:189) as in A49-F3’-TriNKET-Pertuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49MI- F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49MI-F3’-TriNKET-Pertuzumab includes three polypeptides having the sequences of SEQ ID NO:190 (as in A49-F3’-KiH-TriNKET-Pertuzumab), SEQ ID NO:145 (as in A49MI-F3’-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET- Trastuzumab).
[0209] Another exemplary TriNKET of the present disclosure is A49-F3’-KiH- TriNKET-Pertuzumab. A49-F3’-KiH-TriNKET-Pertuzumab includes the same HER2- binding scFv (SEQ ID NO:189) as in A49-F3’-TriNKET-Pertuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the “knob” substitution of T366W. A49-F3’-KiH-TriNKET-Pertuzumab includes three polypeptides, having the sequences of SEQ ID NO:191, SEQ ID NO:147 (as in A49-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET- Trastuzumab). [0210] SEQ ID NO:191 represents the full sequence of the HER2-binding scFv (SEQ ID NO:189) linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv- Fc). The first antibody Fc polypeptide incorporates T366S, L368A, and Y407V substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:191 as described above. Pertuzumab scFv-Fc (KiH) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKCLEWVADVNPNS GGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQ GTLVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:191) [0211] Another exemplary TriNKET of the present disclosure is A49MI-F3’-KiH- TriNKET-Pertuzumab. A49MI-F3’-KiH-TriNKET-Pertuzumab includes the same HER2- binding scFv (SEQ ID NO:189) as in A49-F3’-TriNKET-Pertuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49MI-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide
incorporates the “knob” substitution of T366W. A49MI-F3’-KiH-TriNKET-Pertuzumab includes three polypeptides having the sequences of SEQ ID NO:191 (as in A49-F3’-KiH- TriNKET-Pertuzumab), SEQ ID NO:194 (as in A49MI-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET-Trastuzumab). [0212] Another exemplary TriNKET of the present disclosure is A44-F3’-TriNKET- Pertuzumab. A44-F3’-TriNKET-Pertuzumab includes the same HER2-binding scFv (SEQ ID NO:189) as in A49-F3’-TriNKET-Pertuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A44-F3’- TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A44-F3’-TriNKET-Pertuzumab includes three polypeptides having the sequences of SEQ ID NO:190 (as in A49-F3’-KiH-TriNKET-Pertuzumab), SEQ ID NO:155 (as in A44-F3’-TriNKET-Trastuzumab), and SEQ ID NO:149 (as in A44-F3’-TriNKET- Trastuzumab). [0213] Another exemplary TriNKET of the present disclosure is A44-F3’-KiH- TriNKET-Pertuzumab. A44-F3’-KiH-TriNKET-Pertuzumab includes the same HER2- binding scFv (SEQ ID NO:189) as in A49-F3’-TriNKET-Pertuzumab linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A44-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the “knob” substitution of T366W. A44-F3’-KiH-TriNKET-Pertuzumab includes three polypeptides having the sequences of SEQ ID NO:191 (as in A49-F3’-KiH- TriNKET-Pertuzumab), SEQ ID NO:148 (as in A44-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:149 (as in A44-F3’-TriNKET-Trastuzumab). [0214] Another TriNKET of the present disclosure is A49-F3’-TriNKET-MGAH22. A49-F3’-TriNKET-MGAH22 includes an scFv (SEQ ID NO:171) derived from MGAH22 that binds HER2, linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide linked to the Fab fragment incorporates K360E and K409W substitutions.
A49-F3’-TriNKET-MGAH22 includes three polypeptides having the sequences of SEQ ID NO:192, SEQ ID NO:141 (as in A49-F3’-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET-Trastuzumab). [0215] SEQ ID NO:192 represents the full sequence of the HER2-binding scFv linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv-Fc). The first antibody Fc domain incorporates Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:141 as described above. The scFv (SEQ ID NO:171) includes a heavy chain variable domain of pertuzumab connected to the N-terminus of a light chain variable domain of pertuzumab via a (G
4S)
4 linker (SEQ ID NO:143), the scFv represented as VL-(G
4S)
4-VH (“(G
4S)
4” is represented by SEQ ID NO:143). The heavy and the light variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH, as a result of G100C and G44C substitutions in the VL and VH, respectively. MGAH22 scFv DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGHSPKLLIYSASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYTTPPTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIHWVKQRPEQCLEWIGRIYPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYWGQ GASVTVSSA (SEQ ID NO:171) MGAH22 scFv-Fc DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGHSPKLLIYSASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYTTPPTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIHWVKQRPEQCLEWIGRIYPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYWGQ GASVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:192) [0216] Another TriNKET of the present disclosure is A49MI-F3’-TriNKET-MGAH22. A49MI-F3’-TriNKET-MGAH22 includes the same HER2-binding scFv (SEQ ID NO:171) as in A49-F3’-TriNKET-MGAH22 linked via a hinge including Ala-Ser to a first antibody Fc
polypeptide; and the same NKG2D-binding Fab fragment as in A49MI-F3’-TriNKET- Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49MI- F3’-KiH-TriNKET-MGAH22 includes three polypeptides, having the sequences of SEQ ID NO:192 (as in A49-F3’-TriNKET-MGAH22), SEQ ID NO:145 (as in A49MI-F3’-TriNKET- Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET-Trastuzumab). [0217] Another TriNKET of the present disclosure is A49-F3’-KiH-TriNKET- MGAH22. A49-F3’-KiH-TriNKET-MGAH22 includes the same HER2-binding scFv (SEQ ID NO:171) as in A49-F3’-TriNKET-MGAH22 linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49-F3’- TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide linked to the Fab fragment incorporates the “knob” substitution of T366W. A49-F3’-KiH-TriNKET-MGAH22 includes three polypeptides having the sequences of SEQ ID NO:193, SEQ ID NO:147 (as in A49-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET- Trastuzumab). [0218] SEQ ID NO:193 represents the full sequence of the HER2-binding scFv (SEQ ID NO:171) linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv- Fc). The first antibody Fc polypeptide incorporates T366S, L368A, and Y407V substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:147 as described above. MGAH22 scFv-Fc (KiH) DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGHSPKLLIYSASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYTTPPTFGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIHWVKQRPEQCLEWIGRIYPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYWGQ GASVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:193)
[0219] Another exemplary TriNKET of the present disclosure is A49MI-F3’-KiH- TriNKET-MGAH22. A49MI-F3’-KiH-TriNKET-MGAH22 includes the same HER2- binding scFv (SEQ ID NO:171) as in A49-F3’-TriNKET-MGAH22 linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A49MI-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the “knob” substitution of T366W. A49MI-F3’-KiH-TriNKET-MGAH22 includes three polypeptides having the sequences of SEQ ID NO:193 (as in A49-F3’-KiH- TriNKET-MGAH22), SEQ ID NO:194 (as in A49MI-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:142 (as in A49-F3’-TriNKET-Trastuzumab). [0220] Another exemplary TriNKET of the present disclosure is A44-F3’-TriNKET- MGAH22. A44-F3’-TriNKET-MGAH22 includes the same HER2-binding scFv (SEQ ID NO:171) as in A49-F3’-TriNKET-MGAH22 linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A44-F3’- TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A44-F3’-TriNKET-MGAH22 includes three polypeptides having the sequences of SEQ ID NO:192 (as in A49-F3’-TriNKET-MGAH22), SEQ ID NO:155 (as in A44-F3’-TriNKET-Trastuzumab), and SEQ ID NO:149 (as in A44-F3’-TriNKET- Trastuzumab). [0221] Another exemplary TriNKET of the present disclosure is A44-F3’-KiH- TriNKET-MGAH22. A44-F3’-KiH-TriNKET-MGAH22 includes the same HER2-binding scFv (SEQ ID NO:171) as in A49-F3’-TriNKET-MGAH22 linked via a hinge including Ala- Ser to a first antibody Fc polypeptide; and the same NKG2D-binding Fab fragment as in A44-F3’-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the “knob” substitution of T366W. A44-F3’-KiH-TriNKET-MGAH22 includes three polypeptides having the sequences of SEQ ID NO:193 (as in A49-F3’-KiH-TriNKET- MGAH22), SEQ ID NO:148 (as in A44-F3’-KiH-TriNKET-Trastuzumab), and SEQ ID NO:149 (as in A44-F3’-TriNKET-Trastuzumab).
[0222] In certain embodiments, a TriNKET of the present disclosure is identical to one of the exemplary TriNKETs described above that includes the EW-RVT Fc mutations, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates the substitutions of Q347R, D399V, and F405T, and the antibody Fc polypeptide linked to the HER2-binding scFv incorporates matching substitutions K360E and K409W for forming a heterodimer. In certain embodiments, a TriNKET of the present disclosure is identical to one of the exemplary TriNKETs described above that includes the KiH Fc mutations, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates the “hole” substitutions of T366S, L368A, and Y407V, and the antibody Fc polypeptide linked to the HER2-binding scFv incorporates the “knob” substitution of T366W for forming a heterodimer. [0223] In certain embodiments, a TriNKET of the present disclosure is identical to one of the exemplary TriNKETs described above, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment includes an S354C substitution in the CH3 domain, and the antibody Fc polypeptide linked to the HER2-binding scFv includes a matching Y349C substitution in the CH3 domain for forming a disulfide bond. [0224] As described in International Application No. PCT/US2019/045561, the multi- specific binding proteins disclosed herein are effective in reducing tumor growth and killing cancer cells in in vitro assays and animal models. For example, A49-F3’-TriNKET- Trastuzumab is superior to trastuzumab in inducing NK cell-mediated cytotoxicity against various human cancer cell lines, such as 786-O cells that express low levels of HER2 (HER2+), H661 cells that express moderate levels of HER2 (HER2++), and SkBr3 cells that express high levels of HER2 (HER2+++). Furthermore, the multi-specific binding proteins do not significantly induce NK-mediated killing of healthy non-cancerous human cells (e.g., human cardiomyocytes). [0225] Specific embodiments of a multi-specific binding protein useful in any of the methods of treatment and formulations disclosed herein are described below. [0226] In some embodiments, the first antigen-binding site that binds NKG2D includes a heavy chain variable domain (VH) with complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) having the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively; and a light chain variable domain (VL) with CDR1, CDR2, and CDR3 having the amino acid
sequences of SEQ ID NOs: 99, 100, and 101, respectively. In some embodiments, the first antigen-binding site that binds NKG2D includes a VH with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:94, and a VL with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:98. In some embodiments, the first antigen-binding site that binds NKG2D is a Fab. [0227] In some embodiments, the second antigen-binding site that binds HER2 includes a VH with CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively; and a VL with CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively. In some embodiments, the second antigen-binding site that binds HER2 includes a VH with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:195, and a VL with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:196. In some embodiments, the second antigen-binding site that binds HER2 is a single chain variable fragment (scFv). In some embodiments, the VL of the scFv is linked to the VH of the scFv via a flexible linker. In some embodiments, the flexible linker has the amino acid sequence of SEQ ID NO:143. In some embodiments, the VL of the scFv is positioned to the N- terminus of the VH of the scFv. In some embodiments, the VH of the scFv forms a disulfide bridge with the VL of the scFv (e.g., between residues C44 of the VH of the scFv and C100 of the VL of the scFv). In some embodiments, the scFv has the amino acid sequence of SEQ ID NO:139. [0228] In some embodiments, , the antibody Fc domain comprises a first antibody Fc sequence linked to the Fab that binds NKG2D and a second antibody Fc sequence linked to the scFv that binds HER2. In some embodiments, the first antibody Fc sequence is linked to the heavy chain portion of the Fab. In some embodiments, the scFv is linked to the second antibody Fc sequence via a hinge comprising Ala-Ser. In some embodiments, the first and second antibody Fc sequences each comprise a hinge and a CH2 domain of a human IgG1
antibody. In some embodiments, the first and second antibody Fc sequences each comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody. In some embodiments, the first and second antibody Fc sequences comprise different mutations that promote heterodimerization. In some embodiments, the first antibody Fc sequence is a human IgG1 Fc sequence comprising K360E and K409W substitutions. In some embodiments, the second antibody Fc sequence is a human IgG1 Fc sequence comprising Q347R, D399V, and F405T substitutions. [0229] In some embodiments, the multi-specific binding protein includes a first polypeptide, second polypeptide, and third polypeptide having amino acid sequences of SEQ ID NO:141, SEQ ID NO:140, and SEQ ID NO:142, respectively. Production of Multi-Specific Binding Proteins [0230] The multi-specific binding proteins described above can be made using recombinant DNA technology well known to a skilled person in the art. For example, a first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding the second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding the immunoglobulin light chain can be cloned into a third expression vector; and the first, second, and third expression vectors can be stably transfected together into host cells to produce the multimeric proteins. [0231] A skilled person in the art would appreciate that during production and/or storage of proteins, N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and/or storage). Accordingly, in some embodiments where the N-terminal residue of an amino acid sequence of a polypeptide is E or Q, a corresponding amino acid sequence with the E or Q replaced with pyroglutamate is also contemplated herein. [0232] A skilled person in the art would also appreciate that during protein production and/or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or catalyzed by an enzyme present during production and/or storage). Such removal of K is often observed with proteins that include an Fc domain at their C-termini. Accordingly, in some embodiments where the C-terminal residue of an amino acid sequence of a polypeptide (e.g., an antibody Fc polypeptide) is K, a corresponding amino acid sequence with the K removed is also contemplated herein.
[0233] To achieve the highest yield of the multi-specific binding protein, different ratios of the first, second, and third expression vector can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix. [0234] Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the multi-specific binding protein. The multi-specific binding proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. Pharmaceutical Formulations [0235] The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations that contain a multi-specific binding protein disclosed herein (e.g., A49-F3’-TriNKET-Trastuzumab) at a concentration of greater than 50 mg/mL. The pharmaceutical formulation contains one or more excipients and is maintained at a certain pH. The term “excipient,” as used herein, means any non-therapeutic agent added to the formulation to provide a desired physical or chemical property, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration. Excipients and pH [0236] The one or more excipients in the pharmaceutical composition or pharmaceutical formulation of the present invention contains a buffering agent. The term “buffering agent,” as used herein, refers to one or more components that when added to an aqueous solution is able to protect the solution against variations in pH when adding acid or alkali, or upon dilution with a solvent. In addition to phosphate buffers, glycinate, carbonate, citrate, histidine buffers and the like can be used, in which case, sodium, potassium or ammonium ions can serve as counterion. [0237] In certain embodiments, the buffer or buffer system includes at least one buffer that has a buffering range that overlaps fully or in part with the range of pH 5.5 - 7.4. In certain embodiments, the buffer has a pKa of about 6.0 ± 0.5. In certain embodiments, the buffer contains a histidine buffer. In certain embodiments, the histidine is present at a
concentration of 5 to 100 mM, 10 to 100 mM, 15 to 100 mM, 20 to 100 mM, 5 to 50 mM, 10 to 50 mM, 15 to 100 mM, 20 to 100 mM, 5 to 25 mM, 10 to 25 mM, 15 to 25 mM, 20 to 25 mM, 5 to 20 mM, 10 to 20 mM, or 15 to 20 mM. In certain embodiments, the histidine is present at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 50 mM. In certain embodiments, the histidine is present at a concentration of 20 mM. [0238] The pharmaceutical composition or pharmaceutical formulation disclosed herein may have a pH of 5.5 to 6.5. For example, in certain embodiments, the pharmaceutical composition or pharmaceutical formulation has a pH of 5.5 to 6.5 (i.e., 6.0 ± 0.5), 5.6 to 6.4 (i.e., 6.0 ± 0.4), 5.7 to 6.3 (i.e., 6.0 ± 0.3), 5.8 to 6.2 (i.e., 6.0 ± 0.2), 5.9 to 6.1 (i.e., 6.0 ± 0.1), or 5.95 to 6.05 (i.e., 6.0 ± 0.05). In certain embodiments, the pharmaceutical composition or pharmaceutical formulation has a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation has a pH of 6.0. Under the rules of scientific rounding, a pH greater than or equal to 5.95 and smaller than or equal to 6.05 is rounded as 6.0. [0239] In certain embodiments, the buffer system of the pharmaceutical composition or pharmaceutical formulation contains histidine at 10 to 25 mM, at a pH of 6.0 ± 0.2. In certain embodiments, the buffer system of the pharmaceutical composition or pharmaceutical formulation contains histidine at 20 mM, at a pH of 6.0 ± 0.2. In certain embodiments, the buffer system of the pharmaceutical composition or pharmaceutical formulation contains histidine at 10 to 25 mM, at a pH of 6.0 ± 0.05. In certain embodiments, the buffer system of the pharmaceutical composition or pharmaceutical formulation contains histidine at 20 mM, at a pH of 6.0 ± 0.05. [0240] The one or more excipients in the pharmaceutical composition or pharmaceutical formulation disclosed herein may further contains a sugar or sugar alcohol. Sugars and sugar alcohols are useful in pharmaceutical composition or pharmaceutical formulations as a thermal stabilizer. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains a sugar, for example, a monosaccharide (glucose, xylose, or erythritol), a disaccharide (e.g., sucrose, trehalose, maltose, or galactose), or an oligosaccharide (e.g., stachyose). In specific embodiments, the pharmaceutical composition or pharmaceutical formulation contains sucrose. In certain embodiments, the pharmaceutical composition contains a sugar alcohol, for example, a sugar alcohol derived from a monosaccharide (e.g., mannitol, sorbitol, or xylitol), a sugar alcohol derived from a disaccharide (e.g., lactitol or maltitol), or a sugar alcohol derived from an oligosaccharide. In
specific embodiments, the pharmaceutical composition or pharmaceutical formulation contains sorbitol. [0241] The amount of the sugar or sugar alcohol contained within the formulation can vary depending on the specific circumstances and intended purposes for which the formulation is used. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 50 to 300 mM, 50 to 250 mM, 100 to 300 mM, 100 to 250 mM, 150 to 300 mM, 150 to 250 mM, 200 to 300 mM, 200 to 250 mM, or 250 to 300 mM of the sugar or sugar alcohol. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 250 mM, or 300 mM of the sugar or sugar alcohol. In specific embodiments, the pharmaceutical composition or pharmaceutical formulation contains 250 mM of the sugar or sugar alcohol (e.g., sucrose or sorbitol). [0242] The one or more excipients in the pharmaceutical composition or pharmaceutical formulation disclosed herein further contains a surfactant. The term “surfactant,” as used herein, refers to a surface active molecule containing both a hydrophobic portion (e.g., alkyl chain) and a hydrophilic portion (e.g., carboxyl and carboxylate groups). Surfactants are useful in pharmaceutical composition or pharmaceutical formulations for reducing aggregation of a therapeutic protein. Surfactants suitable for use in the pharmaceutical composition or pharmaceutical formulations are generally non-ionic surfactants and include, but are not limited to, polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); sorbitan esters and derivatives; Triton; sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetadine; lauryl-, myristyl-, linoleyl- or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropylbetaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT
TM series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68 etc.). In certain embodiments, the surfactant is a polysorbate. In certain embodiments, the surfactant is polysorbate 80. [0243] The amount of a non-ionic surfactant contained within the pharmaceutical composition or pharmaceutical formulation of the present invention may vary depending on the specific properties desired of the formulation, as well as the particular circumstances and
purposes for which the formulations are intended to be used. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 0.005% to 0.5%, 0.005% to 0.2%, 0.005% to 0.1%, 0.005% to 0.05%, 0.005% to 0.02%, 0.005% to 0.01%, 0.01% to 0.5%, 0.01% to 0.2%, 0.01% to 0.1%, 0.01% to 0.05%, or 0.01% to 0.02% of the non-ionic surfactant (e.g., polysorbate 80). In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% of the non-ionic surfactant (e.g., polysorbate 80). The concentrations of non-ionic surfactant are provided as % (w/v) values. [0244] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is isotonic. An “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure from about 250 to 350 mOsmol/kgH
2O. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer. In certain embodiments, the osmolarity of the pharmaceutical composition or pharmaceutical formulation is 250 to 350 mOsmol/kgH
2O. In certain embodiments, the osmolarity of the pharmaceutical composition or pharmaceutical formulation is 300 to 350 mOsmol/kgH
2O. [0245] Substances such as a sugar, a sugar alcohol, and NaCl can be included in the pharmaceutical composition or pharmaceutical formulation for desired osmolarity. In certain embodiments, the concentration of NaCl in the pharmaceutical composition or pharmaceutical formulation, if any, is equal to or lower than 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, 0.1 mM, 50 μM, 10 μM, 5 μM, or 1 μM. In certain embodiments, the concentration of NaCl in the pharmaceutical composition or pharmaceutical formulation is below the detection limit. In certain embodiments, no NaCl is added when preparing the pharmaceutical composition or pharmaceutical formulation. [0246] The pharmaceutical composition or pharmaceutical formulation disclosed herein may further include one or more other substances, such as a bulking agent or a preservative. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents. A preservative
reduces bacterial action and may, for example, facilitate the production of a multi-use (multiple-dose) formulation. [0247] The multi-specific binding protein can be formulated in the pharmaceutical composition or pharmaceutical formulation at high concentrations, for example, greater than 50 mg/mL. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains greater than or equal to 60 mg/mL, greater than or equal to 70 mg/mL, greater than or equal to 80 mg/mL, greater than or equal to 90 mg/mL, greater than or equal to 100 mg/mL, greater than or equal to 125 mg/mL, greater than or equal to 150 mg/mL, greater than or equal to 175 mg/mL, or greater than or equal to 200 mg/mL of the multi- specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 60-250 mg/mL, 60-225 mg/mL, 60-200 mg/mL, 60-175 mg/mL, 50-150 mg/mL, 60-150 mg/mL, 60-125 mg/mL, 60-100 mg/mL, 60-90 mg/mL, 60- 80 mg/mL, 60-70 mg/mL, 70-250 mg/mL, 70-225 mg/mL, 70-200 mg/mL, 70-175 mg/mL, 70-150 mg/mL, 70-150 mg/mL, 70-125 mg/mL, 70-100 mg/mL, 70-90 mg/mL, 70-80 mg/mL, 80-250 mg/mL, 80-225 mg/mL, 80-200 mg/mL, 80-175 mg/mL, 80-150 mg/mL, 80- 150 mg/mL, 80-125 mg/mL, 80-100 mg/mL, 80-90 mg/mL, 90-250 mg/mL, 90-225 mg/mL, 90-200 mg/mL, 90-175 mg/mL, 90-150 mg/mL, 90-150 mg/mL, 90-125 mg/mL, 90-100 mg/mL, 100-250 mg/mL, 100-225 mg/mL, 100-200 mg/mL, 100-175 mg/mL, 100-150 mg/mL, 100-125 mg/mL, 125-250 mg/mL, 125-225 mg/mL, 125-200 mg/mL, 125-175 mg/mL, 125-150 mg/mL, 150-250 mg/mL, 150-225 mg/mL, 150-200 mg/mL, 150-175 mg/mL, 175-250 mg/mL, 175-225 mg/mL, 175-200 mg/mL, 200-250 mg/mL, or 200-225 mg/mL of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, or 220 mg/mL of the multi-specific binding protein. Exemplary Formulations [0248] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation of the present invention contains the multi-specific binding protein, histidine, a sugar or sugar alcohol (e.g., sucrose or sorbitol), and a polysorbate (e.g., polysorbate 80), at pH 5.5 to 6.5. [0249] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 10 to 25 mM of
histidine, 200 to 300 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.005% to 0.05% of a polysorbate (e.g., polysorbate 80), at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% of a polysorbate (e.g., polysorbate 80), at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% of a polysorbate (e.g., polysorbate 80), at pH 5.8 to 6.2. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% of a polysorbate (e.g., polysorbate 80), at pH 5.95 to 6.05. [0250] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 10 to 25 mM of histidine, 200 to 300 mM of sucrose, and 0.005% to 0.05% of polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sucrose, and 0.01% of polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sucrose, and 0.01% of polysorbate 80, at pH 5.8 to 6.2. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sucrose, and 0.01% of polysorbate 80, at pH 5.95 to 6.05. [0251] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 10 to 25 mM of histidine, 200 to 300 mM of sorbitol, and 0.005% to 0.05% of polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sorbitol, and 0.01% of polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sorbitol, and 0.01% of polysorbate 80, at pH 5.8 to 6.2. In certain embodiments, the pharmaceutical composition or
pharmaceutical formulation contains 10 to 250 mg/mL of the multi-specific binding protein, 20 mM of histidine, 250 mM of sorbitol, and 0.01% of polysorbate 80, at pH 5.95 to 6.05. [0252] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 50 mg/mL of the multi-specific binding protein, 5 mM to 50 mM of histidine, 50 mM to 300 mM sucrose, and about 0.005% to 0.05% (w/v) of polysorbate 80, at pH 5.5 to 6.5. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 10 to 25 mg/mL of the multi-specific binding protein, 10 mM to 25 mM of histidine, 150 mM to 300 mM sucrose, and about 0.005% to 0.02% (w/v) of polysorbate 80, at pH 5.8 to 6.2. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains about 15 mg/mL of the multi-specific binding protein, about 20 mM of histidine, about 250 mM sucrose, and about 0.01% (w/v) of polysorbate 80, at about pH 6.0. Stability of the Multi-Specific Binding Protein [0253] The pharmaceutical compositions or pharmaceutical formulations disclosed herein exhibit high levels of stability. A pharmaceutical formulation is stable when the multi- specific binding protein within the formulation retains an acceptable degree of physical property, chemical structure, and/or biological function after storage under defined conditions. [0254] Stability can be measured by determining the percentage of the multi-specific binding protein in the formulation that remains in a native conformation after storage for a defined amount of time at a defined temperature. The percentage of a protein in a native conformation can be determined by, for example, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography), where a protein in the native conformation is not aggregated (eluted in a high molecular weight fraction) or degraded (eluted in a low molecular weight fraction). In certain embodiments, more than 95%, 96%, 97%, 98%, or 99% of the multi-specific binding protein has native conformation, as determined by size-exclusion chromatography, after incubation at 4 °C for 3 weeks. In certain embodiments, more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the multi-specific binding protein has native conformation, as determined by size- exclusion chromatography, after incubation at 50 °C for 3 weeks. In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multi-specific binding protein forms a high molecular weight complex (i.e., having a higher molecular weight than the native
protein), as determined by size-exclusion chromatography, after incubation at 4 °C for 3 weeks. In certain embodiments, less than 1%, 2%, 3%, 4%, or 5% of the multi-specific binding protein form a high molecular weight complex (i.e., having a higher molecular weight than the native protein), as determined by size-exclusion chromatography, after incubation at 50 °C for 3 weeks. In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multi-specific binding protein is degraded (i.e., having a lower molecular weight than the native protein), as determined by size-exclusion chromatography, after incubation at 4 °C for 3 weeks. In certain embodiments, less than 1%, 1.5%, 2%, 2.5%, or 3% of the multi-specific binding protein is degraded (i.e., having a lower molecular weight than the native protein), as determined by size-exclusion chromatography, after incubation at 50 °C for 3 weeks. [0255] Stability can also be measured by determining the percentage of multi-specific binding protein present in a more acidic fraction (“acidic form”) relative to the main fraction of protein (“main charge form”). While not wishing to be bound by theory, deamidation of a protein may cause it to become more negatively charged and thus more acidic relative to the non-deamidated protein (see, e.g., Robinson, Protein Deamidation, (2002) PNAS 99(8):5283- 88). The percentage of the acidic form of a protein can be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography) or imaged capillary isoelectric focusing (icIEF). In certain embodiments, at least 50%, 60%, 70%, 80%, or 90% of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation is in the main charge form after incubation at 4 °C for 3 weeks. In certain embodiments, at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation is in the main charge form after incubation at 50 °C for 3 weeks. In certain embodiments, no more than 10%, 20%, 30%, 40%, or 50% of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation is in an acidic form after incubation at 4 °C for 3 weeks. In certain embodiments, no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 85% of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation is in an acidic form after incubation at 50 °C for 3 weeks. [0256] Stability can also be measured by determining the purity of the multi-specific binding protein by electrophoresis after denaturing the protein with sodium dodecyl sulfate (SDS). The protein sample can be denatured in the presence or absence of an agent that
reduces protein disulfide bonds (e.g., β-mercaptoethanol). In certain embodiments, the purity of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol), is at least 95%, 96%, 97%, 98%, or 99% after incubation at 4 °C for 3 weeks. In certain embodiments, the purity of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol), is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after incubation at 50 °C for 3 weeks. In certain embodiments, the purity of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under non-reducing conditions, is at least 95%, 96%, 97%, 98%, or 99% after incubation at 4 °C for 3 weeks. In certain embodiments, the purity of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under non-reducing conditions, is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after incubation at 50 °C for 3 weeks. [0257] Stability can also be measured by determining the parameters of a protein solution by dynamic light scattering. The Z-average and polydispersity index (PDI) values indicate the average diameter of particles in a solution and these measures increase when aggregates are present in the solution. The monomer %Pd value indicates the spread of different monomers detected, where lower values indicate a monodispere solution, which is preferred. The monomer size detected by DLS is useful in confirming that the main population is monomer and to characterize any higher order aggregates that may be present. In certain embodiments, the Z-average value of the pharmaceutical composition or pharmaceutical formulation does not increase by more than 5%, 10%, or 15% after incubation at 4 °C for 3 weeks. In certain embodiments, the Z-average value of the pharmaceutical composition or pharmaceutical formulation does not increase by more than 5%, 10%, 15%, 20%, or 25% after incubation at 50 °C for 3 weeks. In certain embodiments, the PDI value of the pharmaceutical composition or pharmaceutical formulation does not increase by more than 10%, 20%, 30%, 40%, or 50% after incubation at 4 °C for 3 weeks. In certain embodiments, the PDI value of the pharmaceutical composition or pharmaceutical
formulation does not increase by more than 2-fold, 3-fold, 4-fold, or 5-fold after incubation at 50 °C for 3 weeks. [0258] Exemplary methods to determine stability of the multi-specific binding protein in the pharmaceutical composition or pharmaceutical formulation are described in Example 1 of the present disclosure. Additionally, stability of the protein can be assessed by measuring the binding affinity of the multi-specific binding protein to its targets or the biological activity of the multi-specific binding protein in certain in vitro assays, such as the NK cell activation assays and cytotoxicity assays described in WO 2018/152518. Dosage Forms [0259] The pharmaceutical composition or pharmaceutical formulation can be prepared and stored as a liquid formulation or a lyophilized form. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is a liquid formulation for storage at 2-8 °C (e.g., 4 °C) or a frozen formulation for storage at -20 °C or lower. The sugar or sugar alcohol in the formulation is used as a lyoprotectant. [0260] Prior to pharmaceutical use, the pharmaceutical composition or pharmaceutical formulation can be diluted or reconstituted in an aqueous carrier suitable for the route of administration. Other exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. For example, when prepared for intravenous administration, the pharmaceutical composition or pharmaceutical formulation can be diluted in a 0.9% sodium chloride (NaCl) solution, or a 0.9% NaCl solution and 0.01% polysorbate 80. In certain embodiments, the diluted pharmaceutical composition or pharmaceutical formulation is isotonic and suitable for administration by intravenous infusion. [0261] The pharmaceutical composition or pharmaceutical formulation contains the multi-specific binding protein at a concentration suitable for storage. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains the multi-specific binding protein at a concentration of 10-50 mg/mL, 10-40 mg/mL, 10-30 mg/mL, 10-25 mg/mL, 10-20 mg/mL, 10-15 mg/mL, 15-50 mg/mL, 15-40 mg/mL, 15-30 mg/mL, 15-25 mg/mL, 15-20 mg/mL, 20-50 mg/mL, 20-40 mg/mL, 20-30 mg/mL, 20-25 mg/mL, 30-50 mg/mL, 30-40 mg/mL, or 40-50 mg/mL. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains the multi-specific
binding protein at a concentration of 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL. [0262] In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is packaged in a container (e.g., a vial, bag, pen, or syringe). In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the amount of multi-specific binding protein in the container is suitable for administration as a single dose. In certain embodiments, the amount of multi-specific binding protein in the container is suitable for administration in multiple doses. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 0.1 to 2000 mg of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 1 to 2000 mg, 10 to 2000 mg, 20 to 2000 mg, 50 to 2000 mg, 100 to 2000 mg, 200 to 2000 mg, 500 to 2000 mg, 1000 to 2000 mg, 0.1 to 1000 mg, 1 to 1000 mg, 10 to 1000 mg, 20 to 1000 mg, 50 to 1000 mg, 100 to 1000 mg, 200 to 1000 mg, 500 to 1000 mg, 0.1 to 500 mg, 1 to 500 mg, 10 to 500 mg, 20 to 500 mg, 50 to 500 mg, 100 to 500 mg, 200 to 500 mg, 0.1 to 200 mg, 1 to 200 mg, 10 to 200 mg, 20 to 200 mg, 50 to 200 mg, 100 to 200 mg, 0.1 to 100 mg, 1 to 100 mg, 10 to 100 mg, 20 to 100 mg, 50 to 100 mg, 0.1 to 50 mg, 1 to 50 mg, 10 to 50 mg, 20 to 50 mg, 0.1 to 20 mg, 1 to 20 mg, 10 to 20 mg, 0.1 to 10 mg, 1 to 10 mg, or 0.1 to 1 mg of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 0.1 mg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, or 2000 mg of the multi-specific binding protein. Therapeutic Uses Combinational Use with Corticosteroids [0263] In one aspect, the present disclosure provides a method of treating cancer, the method including administering to a subject in need thereof a therapeutically effective amount of a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (administered as a monotherapy or in a combination therapy) and a therapeutically effective amount of a corticosteroid to reduce one or more infusion- related reactions to the multi-specific binding protein, pharmaceutical composition, or
pharmaceutical formulation. The present disclosure also provides a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (administered as a monotherapy or in a combination therapy) for use in a method of treating cancer in combination with a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation. In a combination therapy, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein can be combined with an immunotherapy, such as an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), and/or a chemotherapy, such as a cytoskeletal-disrupting chemotherapeutic agent (e.g., paclitaxel, nab-paclitaxel, docetaxel). [0264] The corticosteroids that are useful in the present invention generally include steroids produced by the adrenocortex, such as glucocorticoids and mineralocorticoids, and synthetic analogs and derivatives of naturally occurring corticosteroids having anti- inflammatory activity. In certain embodiments, the corticosteroid is a glucocorticoid. Glucocorticoids bind the glucocorticoid receptor and reduce inflammation by inhibiting the immune response. In certain embodiments, the corticosteroid is a mineralocorticoid. Mineral corticoids bind the mineralocorticoid receptor and act to regulate Na
+/K
+ concentrations in the serum. Some corticosteroids can have both glucocorticoid and mineralocorticoid functions. Examples of corticosteroids are disclosed in U.S. Patent No. 10,799,599. In certain embodiments, the corticosteroid used in the method disclosed herein is selected from methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, halcinonide, icomethasone, meprednisone, mometasone, rofleponide, RPR 106541, and their respective pharmaceutically acceptable derivatives, such as beclomethasone dipropionate (anhydrous or monohydrate), beclomethasone monopropionate, dexamethasone 21-isonicotinate, fluticasone propionate, icomethasone enbutate, tixocortol 21-pivalate, and triamcinolone acetonide, and pharmaceutically acceptable salts and/or derivatives thereof. [0265] In certain embodiments, the glucocorticoid is methylprednisolone. Exemplary effective amounts of methylprednisolone can be in the range of 8 to 200 mg, 20 to 200 mg,
25 to 200 mg, 50 to 200 mg, 75 to 200 mg, 100 to 200 mg, 125 to 200 mg, 150 to 200 mg, 175 to 200 mg, 25 to 175 mg, 50 to 175 mg, 75 to 175 mg, 100 to 175 mg, 125 to 175 mg, 150 to 175 mg, 20 to 150 mg, 25 to 150 mg, 50 to 150 mg, 75 to 150 mg, 100 to 150 mg, 125 to 150 mg, 25 to 125 mg, 50 to 125 mg, 75 to 125 mg, 100 to 125 mg, 25 to 100 mg, 50 to 100 mg, 75 to 100 mg, 25 to 75 mg, 50 to 75 mg, 25 to 50 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg. In certain embodiments, the effective amount of methylprednisolone is about 125 mg. In certain embodiments, the effective amount of methylprednisolone by oral administration is 8 mg, 16 mg 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg. [0266] In certain embodiments, the glucocorticoid is dexamethasone. Exemplary effective amounts of dexamethasone can be in the range of 8-200 mg, 20-200 mg, 50-200 mg, 100-200 mg, 20-150 mg, 50-150 mg, 50-100 mg, or 100-150 mg. In certain embodiments, the effective amount of dexamethasone by intravenous administration is 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, or 150 mg. In certain embodiments, the effective amount of dexamethasone by oral administration is 8 mg, 16 mg, 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg. [0267] In certain embodiments, the corticosteroid is administered parenterally. In certain embodiments, the corticosteroid is administered intravenously. In certain embodiments, the corticosteroid is administered orally. [0268] The corticosteroid can be administered prior to, simultaneously with, or subsequent to the administration of the multi-specific binding protein. In certain embodiments, the corticosteroid is administered within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multi-specific binding protein (e.g., prior to the beginning of the administration of the multi-specific binding protein). In certain embodiments, the corticosteroid is administered within 1 hour prior to the administration of the multi-specific binding protein (e.g., prior to the beginning of the administration of the multi-specific binding protein). In certain embodiments, the corticosteroid is administered simultaneously with the administration of the multi-specific binding protein. In certain embodiments, the corticosteroid and the multi-specific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the duration of administration of the corticosteroid and the duration of administration of the multi-specific binding protein completely or partially overlap. In certain embodiments, the
corticosteroid is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multi-specific binding protein (e.g., subsequent to the beginning of the administration of the multi-specific binding protein). [0269] In certain embodiments, the corticosteroid is administered on day 1 of the first cycle (i.e., in combination with the first dose of the multi-specific binding protein). In certain embodiments, the corticosteroid is administered only on day 1 of the first cycle (i.e., in combination with the first dose of the multi-specific binding protein). In certain embodiments, the corticosteroid is further administered if an infusion-related reaction persists or recurs. In certain embodiments, infusion-related reactions include a persistent rash, diarrhea, colitis, autoimmune hepatitis, arthritis, glomerulonephritis, cardiomyopathy, or uveitis or another inflammatory eye conditions. [0270] In certain embodiments, the corticosteroid (e.g., methylprednisolone) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. [0271] In certain embodiments, subjects receive premedication treatment including about 125 mg of methylprednisolone administered intravenously within 60 minutes of administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, premedication treatment further includes intravenous or oral administration of 40 to 50 mg diphenhydramine and 800 to 1000 mg of acetaminophen 30 to 60 minutes prior to administration of the multi- specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, about 1.6, 5.2, 10, 15, or 20 mg/kg of multi- specific binding protein (e.g., A49-F3’-TriNKET-Trastuzumab) is administered to the subject after pre-medication treatment. [0272] Exemplary infusion-related reactions to a multi-specific binding protein disclosed herein include cytokine release syndrome, anaphylaxis, chills, fever/pyrexia, hypotension, hypertension, rigors, headache, dizziness, itching, sore throat, laryngeal edema,
angioedema, redness/flushing, rash/urticaria, bronchospasm, tachycardia, bradycardia, auricular fibrillation, hypoxia, respiratory distress/dyspnea/shortness of breath/breathless sensation, chest tightness, nausea, vomiting, pain (e.g., chest pain, back pain), shivering, tremors, myalgia, tiredness, insomnia, asthenia, hypersensitivity, and diarrhea. Clinical presentations of cytokine release syndrome are described in Shimabukuro-Vornhagen et al., include but are not limited to fever (e.g., high fever), fatigue, headache, rash, arthralgia, myalgia, hypotension, vasopressor-requiring circulatory shock, vascular leakage, disseminated intravascular coagulation, and multi-organ system failure. In certain embodiments, the co-administration of the corticosteroid reduces one or more of the infusion- related reactions in the subject. Combinational Use with Antihistamines [0273] An antihistamine can be used to avoid or mitigate an allergic response (e.g., anaphylaxis) to the multi-specific binding protein. Accordingly, in certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an antihistamine. Exemplary antihistamines are disclosed in U.S. Patent No. 10,898,693. In certain embodiments, the antihistamine used in the method disclosed herein is selected from crivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, cyclizine, chlorpheniramine, chlorodiphenhydramine, clemastine, cromolyn, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, nedocromil, olopatadine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, rupatadine, tripelennamine, triprolidine, and combinations thereof. In certain embodiments, the antihistamine is diphenhydramine. In certain embodiments, the therapeutically effective amount of diphenhydramine is 10 to100 mg, 20 to100 mg, 30 to100 mg, 40 to100 mg, 50 to100 mg, 60 to 100 mg, 70 to 100 mg, 80 to 100 mg, 90 to 100 mg, 10 to 90 mg, 20 to 90 mg, 30 to 90 mg, 40 to 90 mg, 50 to 90 mg, 60 to 90 mg, 70 to 90 mg, 80 to 90 mg, 10 to 80 mg, 20 to 80 mg, 30 to 80 mg, 40 to 80 mg, 50 to 80 mg, 60 to 80 mg, 70 to 80 mg, 10 to 70 mg, 20 to 70 mg, 30 to 70 mg, 40 to 70 mg, 50 to 70 mg, 60 to 70 mg, 10 to 60 mg, 20 to 60 mg, 30 to 60 mg, 40 to 60 mg, 50 to 60 mg, 10 to 50 mg, 20 to 50 mg, 30 to 50 mg, 40 to 50 mg, 20 to 40 mg, 30 to 40 mg, 20 to 30 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg. In certain embodiments, the therapeutically effective amount of diphenhydramine is 40 to 50 mg.
[0274] In certain embodiments, the antihistamine is administered parenterally. In certain embodiments, the antihistamine is administered intravenously. In certain embodiments, the antihistamine is administered orally. [0275] The antihistamine can be administered prior to, simultaneously with, or subsequent to the administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the antihistamine is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multi-specific binding protein (e.g., prior to the beginning of the administration of the multi-specific binding protein), pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the antihistamine is administered with every dose of the multi-specific binding protein. [0276] In certain embodiments, the antihistamine (e.g., diphenhydramine) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. [0277] In certain embodiments, a subject receives premedication treatment including 40 to 50 mg of diphenhydramine administered intravenously or orally 30 to 60 minutes prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, premedication treatment further includes intravenous or oral administration of 800 to 100 mg acetaminophen prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. [0278] Where the method of treatment disclosed herein includes multiple doses (e.g., five or more doses) of the multi-specific binding protein, in certain embodiments, the antihistamine is administered with the first dose, the first two doses, the first three doses, the first four doses, or the first five doses of the multi-specific binding protein.
Combinational Use with Analgesics and Antipyretics [0279] An analgesic can be used to relieve pain as a result of the administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein, whether administered as a monotherapy or as a combination therapy with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab) or a cytoskeletal-disrupting chemotherapeutic agent. Accordingly, in certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an analgesic. Exemplary analgesics are disclosed in U.S. Patent Application Publication No. 2015/0342989 and U.S. Patent No. 10,899,834. In certain embodiments, the analgesic used in the method disclosed herein is selected from acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, etoricoxib, codeine, dihydrocodeine, morphine or a morphine derivative or pharmaceutically acceptable salt thereof, diacetylmorphine, hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene, and pentazocine, and pharmaceutically acceptable salts thereof. In certain embodiments, the analgesic is acetaminophen. In certain embodiments, the therapeutically effective amount of acetaminophen is in the range of 325-1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. [0280] In certain embodiments, the analgesic is administered parenterally. In certain embodiments, the analgesic is administered intravenously. In certain embodiments, the analgesic is administered orally.
[0281] The analgesic can be administered prior to, simultaneously with, or subsequent to the administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the analgesic is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multi- specific binding protein (e.g., prior to the beginning of the administration of the multi- specific binding protein). In certain embodiments, the analgesic is administered simultaneously with the administration of the multi-specific binding protein. In certain embodiments, the analgesic and the multi-specific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the analgesic (e.g., acetaminophen) is administered with every dose of the multi-specific binding protein. [0282] In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. [0283] In certain embodiments, the duration of administration of the analgesic and the duration of administration of the multi-specific binding protein completely or partially overlap. In certain embodiments, the analgesic is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multi-specific binding protein (e.g., subsequent to the beginning of the administration of the multi-specific binding protein). [0284] An antipyretic can be used to prevent or reduce fever as a result of the administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein, whether administered as a monotherapy or as a combination therapy with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab) or a cytoskeletal-disrupting chemotherapeutic agent. Accordingly, in certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an antipyretic. Exemplary antipyretics are disclosed in U.S. Patent Application Publication No. 2015/0342989. In certain embodiments, the antipyretic used in the method disclosed herein is selected from acetaminophen, salicylamide, salicyl salicylate, methyl salicylate,
magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, and etoricoxib. In certain embodiments, the antipyretic is acetaminophen. In certain embodiments, the therapeutically effective amount of acetaminophen is in the range of 325-1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. [0285] In certain embodiments, the antipyretic is administered parenterally. In certain embodiments, the antipyretic is administered intravenously. In certain embodiments, the antipyretic is administered orally. [0286] The antipyretic can be administered prior to, simultaneously with, or subsequent to the administration of the multi-specific binding protein. In certain embodiments, the antipyretic is administered within 2 hours, 1.5 hours, 1 hour (60 minutes), 45 minutes, 30 minutes, or 15 minutes prior to the administration of the multi-specific binding protein (e.g., prior to the beginning of the administration of the multi-specific binding protein). In certain embodiments, the antipyretic is administered simultaneously with the administration of the multi-specific binding protein. In certain embodiments, the antipyretic and the multi-specific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the duration of administration of the antipyretic and the duration of administration of the multi-specific binding protein completely or partially overlap. In certain embodiments, the antipyretic is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multi-specific binding protein (e.g., subsequent to the beginning of the administration of the multi-specific binding protein). In
certain embodiments, the antipyretic (e.g., acetaminophen) is administered with every dose of the multi-specific binding protein. [0287] The antipyretic can be administered prior to, simultaneously with, or subsequent to the administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. [0288] In certain embodiments, subjects receive premedication treatment including 800 to 1000 mg of acetaminophen administered intravenously or orally 30 to 60 minutes prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed. In certain embodiments, premedication treatment further includes intravenous or oral administration of 40 to 50 mg diphenhydramine prior to administration of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed. Dosage Regimens [0289] In certain embodiments, the method includes administering to a subject in need thereof a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3’-TriNKET-Trastuzumab) in an initial four-week treatment cycle on day 1, day 8, and day 15. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered to the subject only on these three days in the initial four-week treatment cycle. In specific embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is not administered to the subject on day 22. This regimen is a dose intensification schedule, which is designed to reach maximal saturation of the target as early as possible during the course of the treatment while minimizing the infusion burden for the patient.
[0290] In certain embodiments, the method further includes administering to the subject the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation on Day 1 and Day 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered to the subject only on these two days in each subsequent four-week treatment cycle. In specific embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is not administered to the subject on day 8 or day 22. The subsequent treatment cycles, in which the subject receives administration of the multi- specific binding protein, pharmaceutical composition, or pharmaceutical formulation once every two weeks, are designed to maintain a certain level of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation in the subject. In certain embodiments, the subject receives at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 subsequent treatment cycles. In certain embodiments, the subject receives subsequent treatment cycles until regression of the cancer. [0291] In certain embodiments, the method includes administering to a subject in need thereof a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3’-TriNKET-Trastuzumab) as a monotherapy. In certain embodiments, the method includes administering the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3’- TriNKET-Trastuzumab) intravenously as a one hour infusion in four-week treatment cycles. [0292] In certain embodiments, the method includes administering to a subject in need thereof a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3’-TriNKET-Trastuzumab) over a 45 to 75 min., 50 to 75 min., 55 to 75 min., 60 to 75 min., 65 to 75 min., 70 to 75 min., 45 to 70 min., 50 to 70 min., 55 to 70 min., 60 to 70 min., 65 to 70 min., 45 to 65 min., 50 to 65 min., 55 to 65 min., 60 to 65 min., 45 to 60 min., 50 to 60 min., 55 to 60 min., 45 to 135 min., 60 to 135 min., 75 to 135 min., 90 to 135 min., 105 to 135 min., 120 to 135 min., 45 to 120 min., 60 to 120 min., 75 to 120 min., 90 to 120 min., 105 to 120 min., 45 to 105 min., 60 to 105 min., 75 to 105 min., 90 to 105 min., 45 to 90 min., 60 to 90 min., 75 to 90 min., 45 to 75 min., 60 to 75 min., 45 to 60 min., about 45 min., about 50 min., about 55 min., about 60 min., about 65 min., about 70 min., about 75 min., about 90 min., about 105 min., or about 120 min. period.
[0293] In certain embodiments, one or more doses of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles contain 0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-5 mg/kg, 0.1-2 mg/kg, 0.1-1 mg/kg, 0.1-0.5 mg/kg, 0.1-0.2 mg/kg, 0.2-20 mg/kg, 0.2-10 mg/kg, 0.2-5 mg/kg, 0.2-2 mg/kg, 0.2-1 mg/kg, 0.2-0.5 mg/kg, 0.5-20 mg/kg, 0.5-10 mg/kg, 0.5-5 mg/kg, 0.5-2 mg/kg, 0.5-1 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, or 1-2 mg/kg of the multi-specific binding protein relative to the body weight of the subject. In certain embodiments, one or more doses of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles contain 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg of the multi-specific binding protein relative to the body weight of the subject. [0294] In certain embodiments, each of the doses of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles contain 0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-5 mg/kg, 0.1-2 mg/kg, 0.1-1 mg/kg, 0.1-0.5 mg/kg, 0.1-0.2 mg/kg, 0.2-20 mg/kg, 0.2-10 mg/kg, 0.2-5 mg/kg, 0.2-2 mg/kg, 0.2-1 mg/kg, 0.2-0.5 mg/kg, 0.5-20 mg/kg, 0.5-10 mg/kg, 0.5-5 mg/kg, 0.5-2 mg/kg, 0.5-1 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, or 1-2 mg/kg of the multi-specific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses of the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles contain a same amount in the range of 0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-5 mg/kg, 0.1-2 mg/kg, 0.1-1 mg/kg, 0.1-0.5 mg/kg, 0.1- 0.2 mg/kg, 0.2-20 mg/kg, 0.2-10 mg/kg, 0.2-5 mg/kg, 0.2-2 mg/kg, 0.2-1 mg/kg, 0.2-0.5 mg/kg, 0.5-20 mg/kg, 0.5-10 mg/kg, 0.5-5 mg/kg, 0.5-2 mg/kg, 0.5-1 mg/kg, 1-20 mg/kg, 1- 10 mg/kg, 1-5 mg/kg, or 1-2 mg/kg of the multi-specific binding protein relative to the body weight of the subject. [0295] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contain 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg of the
multi-specific binding protein. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg of the multi-specific binding protein. [0296] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 5.2 × 10
-5 mg/kg, 1.6 × 10
-4 mg/kg, 5.2 × 10
-4 mg/kg, 1.6 × 10
-3 mg/kg, 5.2 × 10
-3 mg/kg, 1.6 × 10
-2 mg/kg, 5.2 × 10
-2 mg/kg, 1.6 × 10
-1 mg/kg, 0.52 mg/kg, 1.6 mg/kg, 5.2 mg/kg, 10 mg/kg, or 20 mg/kg of the multi-specific binding protein. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 5.2 × 10
-5 mg/kg, 1.6 × 10
-4 mg/kg, 5.2 × 10
-4 mg/kg, 1.6 × 10
-3 mg/kg, 5.2 × 10
-3 mg/kg, 1.6 × 10
-2 mg/kg, 5.2 × 10
-2 mg/kg, 1.6 × 10
-1 mg/kg, 0.52 mg/kg, 1 mg/kg, 1.6 mg/kg, 5.2 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg of the multi-specific binding protein. [0297] In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered intravenously. For example, in certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by intravenous infusion, e.g., with a prefilled bag, a prefilled pen, or a prefilled syringe. In certain embodiments, the multi-specific binding protein, in a pharmaceutical composition or pharmaceutical formulation disclosed herein, is diluted prior to administration. For example, in certain embodiments, the pharmaceutical composition or pharmaceutical formulation is diluted with sodium chloride and is administered intravenously from a 250 ml saline bag. The intravenous infusion may be for about one hour (e.g., 50 to 80 minutes). In certain embodiments, the bag is connected to a channel including a tube and/or a needle. [0298] In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered subcutaneously. For example, in certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by subcutaneous injection using a syringe or an auto-injector. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by subcutaneous injection at the abdomen, arm, or thigh. In specific embodiments, the pharmaceutical composition or pharmaceutical formulation suitable for subcutaneous administration contains the multi-
specific binding protein at a concentration greater than 50 mg/mL as described in the Pharmaceutical Formulations subsection above. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains greater than or equal to 60 mg/mL, greater than or equal to 70 mg/mL, greater than or equal to 80 mg/mL, greater than or equal to 90 mg/mL, greater than or equal to 100 mg/mL, greater than or equal to 125 mg/mL, greater than or equal to 150 mg/mL, greater than or equal to 175 mg/mL, or greater than or equal to 200 mg/mL of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 60-250 mg/mL, 60-225 mg/mL, 60-200 mg/mL, 60-175 mg/mL, 50-150 mg/mL, 60-150 mg/mL, 60-125 mg/mL, 60- 100 mg/mL, 60-90 mg/mL, 60-80 mg/mL, 60-70 mg/mL, 70-250 mg/mL, 70-225 mg/mL, 70-200 mg/mL, 70-175 mg/mL, 70-150 mg/mL, 70-150 mg/mL, 70-125 mg/mL, 70-100 mg/mL, 70-90 mg/mL, 70-80 mg/mL, 80-250 mg/mL, 80-225 mg/mL, 80-200 mg/mL, 80- 175 mg/mL, 80-150 mg/mL, 80-150 mg/mL, 80-125 mg/mL, 80-100 mg/mL, 80-90 mg/mL, 90-250 mg/mL, 90-225 mg/mL, 90-200 mg/mL, 90-175 mg/mL, 90-150 mg/mL, 90-150 mg/mL, 90-125 mg/mL, 90-100 mg/mL, 100-250 mg/mL, 100-225 mg/mL, 100-200 mg/mL, 100-175 mg/mL, 100-150 mg/mL, 100-125 mg/mL, 125-250 mg/mL, 125-225 mg/mL, 125- 200 mg/mL, 125-175 mg/mL, 125-150 mg/mL, 150-250 mg/mL, 150-225 mg/mL, 150-200 mg/mL, 150-175 mg/mL, 175-250 mg/mL, 175-225 mg/mL, 175-200 mg/mL, 200-250 mg/mL, or 200-225 mg/mL of the multi-specific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation contains 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, or 220 mg/mL of the multi-specific binding protein. Cancers Suitable for Treatment [0299] The multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein can be used to treat various types of cancer. In certain embodiments, the cancer is a solid tumor. In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, or uterine cancer. In yet other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous
melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin- secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor. [0300] In certain embodiments, the cancer is a hematologic malignancy. In certain embodiments, the hematologic malignancy is leukemia. In certain embodiments, the
leukemia is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. [0301] In specific embodiments, the types of cancer that can be treated with the multi- specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein, where the second antigen-binding site of the multi-specific binding protein binds HER2, include but are not limited to breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, and gallbladder cancer. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a locally advanced or metastatic solid tumor. In certain embodiments, the cancer is gastric cancer (e.g., HER2 high or HER23+). In certain embodiments, the cancer is urothelial bladder cancer (e.g., urothelial bladder cancer expressing HER2). In certain embodiments, the cancer is metastatic breast cancer (e.g., metastatic triple negative breast cancer). In certain embodiments, the cancer is a solid tumor (e.g., HER2 high or HER23+). [0302] Disclosed herein, in various embodiments, is a method of treating a HER2- overexpressing gastric cancer in a subject in need thereof, including administering an effective amount of a multi-specific binding protein or a pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., determined by in situ hybridization). [0303] Also disclosed herein, in various embodiments, is a method of treating a HER2- overexpressing gastric cancer in a subject in need thereof, including administering an anti- PD-1 antibody (e.g., nivolumab) and an effective amount of a multi-specific binding protein or a pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., determined by in situ hybridization).
[0304] Also disclosed herein, in various embodiments, is a method of treating a HER2- overexpressing gastric cancer in a subject in need thereof, including administering nab- paclitaxel and an effective amount of a multi-specific binding protein or a pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., determined by in situ hybridization). [0305] Also disclosed herein, in various embodiments, is a method of treating a HER2- overexpressing gastric cancer in a subject in need thereof, including administering (e.g. via intravenous infusion) an effective amount of a multi-specific binding protein or a pharmaceutical formulation disclosed herein, in combination with nivolumab, and further in combination with nab-paclitaxel. In certain embodiments, the gastric cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the gastric cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., determined by in situ hybridization). [0306] Disclosed herein, in various embodiments, is a method of treating metastatic triple negative breast cancer (TNBC) in a subject in need thereof, including administering an effective amount of a multi-specific binding protein or a pharmaceutical formulation disclosed herein. [0307] In certain embodiments, the subject treated by the method disclosed herein has a HER2-positive cancer. Methods of determining HER2 expression in a cancer include but are not limited to immunohistochemistry (IHC). Anti-HER2 antibodies (e.g., Ventana 4B5 antibody and Bond Oracle CB11 antibody) have been approved by the FDA for detecting HER2, and immunohistochemistry kits (e.g., HercepTest
TM) are commercially available. The level of HER2 expression in a tumor sample, as detected by immunohistochemistry, can be quantified and scored as 1+, 2+, or 3+ according to the ASCO/CAP guideline (Wolff et al., (2007) J. Clin. Oncol. 25(1):118-45) and the 2018 update (Wolff et al., (2018) J. Clin. Oncol. 36(20):2105-22). Under the 2018 ASCO/CAP guideline, a cancer or tumor is scored as HER2 3+ if in a sample, circumferential membrane staining of HER2 is complete, intense and in >10% of tumor cells, which is readily appreciated using a low power objective and observed within a homogeneous and contiguous invasive cell population. A cancer or tumor is scored as HER22+ if weak to moderate complete membrane staining of HER2 is observed
in >10% of tumor cells in a sample. A cancer or tumor is scored as HER21+ if incomplete membrane staining of HER2 is faint or barely perceptible and in >10% of tumor cells in a sample. A cancer or tumor is scored as HER2 negative if no HER2 staining is observed or membrane staining is incomplete and is faint or barely perceptible and in ≤10% of tumor cells. Where a cancer or tumor is scored as HER22+ in this initial IHC assessment, a reflex test (same specimen using ISH) or a new test (new specimen if available, using IHC or ISH) must be ordered. Based on the result of the reflex test or the new test, the cancer or tumor may be re-scored as HER23+ or 1+. [0308] It is understood that ERBB2 gene amplification is generally correlated with HER2 overexpression. Determining whether ERBB2 gene is amplified in a cancer tissue sample may help reduce false-positive results from immunohistochemistry of the same sample (see, e.g., Sarode et al., (2015) Arch. Pathol. Lab. Med. 139:922–28). Accordingly, in certain embodiments, the cancer or tumor in the subject has been assessed by ERBB2 gene amplification. In certain embodiments, the cancer or tumor harbors ERBB2 gene amplification, for example, an average ERBB2 gene copy number greater than or equal to 4.0 signals per cell (e.g., greater than or equal to 6.0 signals per cell). In certain embodiments, the cancer or tumor (e.g., a HER21+ cancer or tumor) has an average ERBB2 gene copy number less than 4.0 signals per cell. In certain embodiments, the cancer or tumor (e.g., a HER2 1+ cancer or tumor) does not have ERBB2 gene amplification. Methods of detecting gene amplification include but are not limited to fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), quantitative PCR, and DNA sequencing. In certain embodiments, ERBB2 gene amplification is determined by FISH. In certain embodiments, ERBB2 gene amplification is determined by DNA sequencing (e.g., deep sequencing). [0309] New technologies can be employed to assess HER2 levels in patient samples. For example, the automated quantitative analysis technology can quantitatively assess HER2 expression by measuring the intensity of antibody-conjugated fluorophores. The HERmark technology measures HER2 expression through a proximity-based release of antibody-bound fluorescent tags. The quantitative IHC technology converts antibody/antigen complexes into red dots, subsequently counted to quantify HER2 expression. The time-resolved fluorescence energy transfer technology enables assessment of HER2 expression through the detection of fluorescence emitted by two fluorophores in close proximity. The quantitative real-time polymerase chain reaction technology enables quantitative measurement of the amount of
HER2 mRNA in a sample. The flow cytometry technology enables measurement of the number of HER2 proteins on the surface of a cell. These assays can complement the results of the IHC or FISH assays, thereby obtaining more accurate assessment of the HER2 level in the cancer or tumor. [0310] For example, in certain embodiments, the cancer or tumor can be assessed by flow cytometry. As described in Example 5 below, Molecules of Equivalent Soluble Fluorochrome (MESF) beads with manufacturer predetermined fluorophore amounts can be used to generate a calibration curve. This calibration curve can be used to correlate the geometric mean fluorescence intensity (MFI) of a given cell population to standardized molecules numbers. The reagent detecting the HER2 proteins can be a protein that binds HER2 (e.g., a HER2 TriNKET disclosed herein) coupled with a fluorophore. Where the sample is a cell line, the geometric mean of the number of HER2 proteins on the cells can be determined. Cell lines with determined levels of HER2 can then be used as references, e.g., in an IHC assay, to determine the level of HER2 in tumor samples. In fact, SKBR3, MDA- MB-175 and MDA-MB-231 cell lines are used as references to set level 3, level 1 and level 0 staining intensity for the HercepTest. In certain embodiments, a cell line having a geometric mean of 500,000 or more (e.g., 600,000 or more, 700,000 or more, 800,000 or more, or 900,000 or more) HER2 proteins on each cell corresponds to the cells that have complete, intense circumferential membrane staining of HER2 in a HER23+ sample; a cell line having a geometric mean of 75,000 to 499,999 (e.g., 80,000 to 499,999, 90,000 to 499,999, 100,000 to 499,999, 110,000 to 499,999, 120,000 to 499,999, 85,000 to 399,999, 90,000 to 399,999, 100,000 to 399,999, 110,000 to 399,999, 115,000 to 399,999, 120,000 to 399,999, 85,000 to 299,999, 90,000 to 299,999, 95,000 to 299,999, 100,000 to 299,999, 105,000 to 299,999, 110,000 to 299,999, 85,000 to 199,999, 90,000 to 199,999, 95,000 to 199,999, 100,000 to 199,999, 105,000 to 199,999, or 110,000 to 199,999,) HER2 proteins on each cell corresponds to the cells that have weak to moderate complete membrane staining of HER2 in a HER22+ sample; a cell line having a geometric mean of 10,000 to 74,999 (e.g., 11,000 to 74,999, 11,000 to 69,999, 11,000 to 64,999, 11,000 to 59,999, or 11,000 to 54,999) HER2 proteins on each cell corresponds to the cells that have faint or barely perceptible, incomplete membrane staining of HER2 in a HER21+ sample. A cell line having a geometric mean of less than 10,000 (e.g., 9000 or less, 8000 or less, 7000 or less, 6000 or less, 5000 or less, 4000 or less) HER2 proteins on each cell corresponds to negative membrane staining in a
HER2 0 sample. According to the 2018 ASCO/CAP guideline, more than 10% of tumor cells in a sample must meet the required HER2 level threshold. [0311] In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 1+, 2+, or 3+. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 1+ or 2+. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 2+ or 3+. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 1+. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 2+. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor (e.g., any one of the types of cancer disclosed in the preceding three paragraphs) with HER2 level scored as 3+. In certain embodiments, the HER2 level is determined by immunohistochemistry (e.g., HercepTest
TM). In certain embodiments, the subject treated by the method disclosed herein has a tumor that shows HER2 expression at least as a faint/barely perceptible membrane staining detected in at least or more than 10% of the tumor cells. In certain embodiments, the subject treated by the method disclosed herein has a tumor that shows HER2 expression at least as a weak to moderate complete membrane staining detected in at least or more than 10% of the tumor cells. In certain embodiments, the subject treated by the method disclosed herein has a tumor that shows HER2 expression at least as a strong complete membrane staining detected in at least or more than 10% of the tumor cells. [0312] In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, uterine cancer, a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral
lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin- secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is a hematologic malignancy
such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, or metastatic breast cancer. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple negative breast cancer with a HER2 level scored as 1+. In certain embodiments, the cancer or tumor is positive for a hormone receptor (i.e., positive for estrogen receptor, progesterone receptor, or both) and negative for HER2 (e.g., as assessed by IHC). According to the 2018 ASCO/CAP HER2 testing guideline update, breast cancer is considered HER2 negative in cases of IHC 0 and 1+ results, or IHC 2+ with a negative ISH assay. Thus, in certain embodiments, the cancer or tumor is a triple negative breast cancer negative for estrogen receptor and progesterone receptor (e.g., as assessed by IHC) with a HER2 level scored as IHC 1+. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., as assessed by IHC) with a HER2 level scored as IHC 1+. In certain embodiments, the HER2 1+ cancer or tumor has an average ERBB2 gene copy number less than 4.0 signals per cell. In certain embodiments, the HER21+ cancer or tumor does not have ERBB2 gene amplification. [0313] In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 2+, and the cancer or tumor is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, uterine cancer, a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral
lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin- secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 2+, and the cancer or tumor is a hematologic malignancy
such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 2+, and the cancer or tumor is breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, or metastatic breast cancer. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored as 2+, and the cancer or tumor is a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple negative breast cancer with a HER2 level scored as IHC 2+ with a negative ISH assay. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., as assessed by IHC) with a HER2 level scored as IHC 2+ with a negative ISH assay. In certain embodiments, the HER22+ cancer or tumor has an average ERBB2 gene copy number less than 4.0 signals per cell. In certain embodiments, the HER22+ cancer or tumor does not have ERBB2 gene amplification. [0314] In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level that is not high (i.e., scored as 1+ or 2+), and the cancer or tumor is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, uterine cancer, a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma/carcinoma, chronic lymphocytic leukemia,
chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level that is not high (i.e., scored as 1+ or 2+), and the cancer or tumor is a hematologic malignancy such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T- lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored that is not high (e.g., scored as 1+ or 2+), and the cancer or tumor is breast cancer, thyroid cancer,
gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous carcinoma of the skin, carcinoma of the salivary gland, biliary tract cancer, lung squamous, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, and metastatic breast cancer. In certain embodiments, the subject treated by the method disclosed herein has a cancer or tumor with a HER2 level scored that is not high (i.e., scored as 1+ or 2+), and the cancer or tumor is a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple negative breast cancer with a HER2 level scored as IHC 1+, or IHC 2+ with a negative ISH assay. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., as assessed by IHC) with a HER2 level scored as IHC 1+, or IHC 2+ with a negative ISH assay. In certain embodiments, the cancer or tumor with a HER2 level score that is not high (i.e., scored as 1+ or 2+) has an average ERBB2 gene copy number less than 4.0 signals per cell. In certain embodiments, the cancer or tumor with a HER2 level score that is not high (i.e., scored as 1+ or 2+) does not have ERBB2 gene amplification. [0315] In certain embodiments, the subject treated in accordance with the methods disclosed herein has not received prior therapy for treating the cancer. In certain embodiments, the subject treated in accordance with the methods disclosed herein has not received prior chemotherapy or immunotherapy for treating the cancer. In certain embodiments, the subject treated in accordance with the methods disclosed herein has received a prior therapy (e.g., a chemotherapy or immunotherapy) but continues to experience cancer progression despite the prior therapy. In certain embodiments, the subject treated in accordance with the methods disclosed herein has experienced cancer regression after receiving a prior therapy (e.g., a chemotherapy or immunotherapy), but later experienced cancer relapse. In certain embodiments, the subject treated in accordance with the methods disclosed herein is intolerant to a prior therapy (e.g., a chemotherapy or immunotherapy). [0316] In certain embodiments, the subject treated in accordance with the methods disclosed herein meets one or more of the inclusion criteria of a clinical trial cohort (e.g., the accelerated titration cohort, the “3+3” dose escalation cohort, the safety/PK/PD expansion cohorts, the urothelial bladder cancer (UBC) cohort, the metastatic breast cancer (MBC) cohort, the Basket solid tumors with high HER2 expression (HER23+) cohort, or the
Combination therapy with pembrolizumab cohort) described in Example 3. In certain embodiments, the subject treated in accordance with the methods disclosed herein meets all the inclusion criteria of a clinical trial cohort (e.g., the accelerated titration cohort, the “3+3” dose escalation cohort, the safety/PK/PD expansion cohorts, the UBC cohort, the MBC cohort, the Basket solid tumors with high HER2 expression (HER23+) cohort, or the Combination therapy with pembrolizumab cohort) described in Example 3. [0317] In certain embodiments, provide herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - histologically or cytologically proven locally advanced or metastatic solid tumors, for which no standard therapy exists, or standard therapy has failed; - primary tumors having documented HER2 expression detected by immunohistochemistry; - detectable erbb2 amplification and/or erbb2 activating mutations in a tumor biopsy; - histologically or cytologically proven locally advanced or metastatic solid tumors for which no standard therapy exists, or standard therapy has failed; - has HER2-expressing tumors (at least 1+ at the time of screening) as determined by immunohistochemistry using a CLIA accredited (or equivalent) method; - has disease measurable with at least one unidimensional measurable lesion by RECIST 1.1; - ECOG performance status of 0 or 1 at study entry and an estimated life expectancy of at least 3 months; - baseline left ventricular ejection fraction (LVEF) ≥ 55% as measured by echocardiography or multigated acquisition (MUGA) scan; - adequate hematological function defined by white blood cell (WBC) count ≥ 3 × 10
9/L with absolute neutrophil count (ANC) ≥ 1.5 × 10
9/L, lymphocyte count ≥ 0.5 × 10
9/L, platelet count ≥ 75 × 10
9/L, and hemoglobin ≥ 9 g/dL (may have been transfused); - adequate hepatic function defined by a total bilirubin level ≤ 1.5 × the upper limit of normal (ULN), an aspartate aminotransferase (AST) level ≤ 2.5 × ULN, and an
alanine aminotransferase (ALT) level ≤ 2.5 × ULN or, for patients with documented metastatic disease to the liver, AST and ALT levels ≤ 5 × ULN; and/or - adequate renal function defined by an estimated creatinine clearance > 50 mL/min according to the Cockcroft-Gault formula. [0318] In certain embodiments, provide herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - is eligible to receive nivolumab per its label for a malignancy of epithelial origin; or has a tumor that does not have a standard therapy or standard therapy has failed; and - has received anti-PD-1 or anti-PD-L1 as a previous line of therapy; or has not received anti-PD-1 or anti-PD-L1 as a previous line of therapy and has experienced a grade 3 or 4 drug-related toxicity or a grade 2 drug related toxicity impacting the lungs or the neurological system, related to the prior anti-PD-1 or anti-PD-L1 therapy. [0319] In certain embodiments, provide herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - is eligible to receive nab-paclitaxel per its label after failure of combination therapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy, and has not been exposed to taxanes within the last six months; - has a tumor that has no standard therapy or standard therapy has failed; or - has an advanced (unresectable/recurrent/metastatic) triple negative breast cancer (TNBC). [0320] In certain embodiments, provide herein are methods of treating a subject having urothelial bladder cancer (UBC) and one or more (e.g., all) of the following characteristics: - histologically or cytologically documented locally advanced or metastatic transitional cell carcinoma of the urothelium (including renal pelvis, ureters, urinary urothelial, urethra); - radiographic disease progression after the subject’s last line of therapy; - received treatment with one (and no more than one) platinum-containing regimen (e.g., platinum plus another agent such as gemcitabine, methotrexate, vinblastine, doxorubicin, etc.) for inoperable locally advanced or metastatic urothelial carcinoma with radiographic progression or recurrence within 6 months after the last
administration of a platinum-containing regimen as an adjuvant, which would be considered failure of a first-line, platinum-containing regimen; - treatment with a checkpoint inhibitor (i.e. anti-PD-1 or anti-PD-L1), with radiographic progression (optionally, treatment with a combination of platinum-based therapy with PD-1/PD-L1-based therapy); and/or - HER2 expression of at least 1+ determined by immunohistochemistry. [0321] In certain embodiments, provided herein are methods for treating a subject having metastatic breast cancer (MBC) and one or more (e.g., all) of the following characteristics: - histologically confirmed MBC; - no more than 3 prior lines of cytotoxic therapy for metastatic disease; - previous received treatment with taxane and an anthracycline, unless anthracycline is contraindicated; - a tumor scoring 1+ or 2+ by IHC, and if scoring is 2+, and the existence of tumor amplification of ERRB2 must be ruled by an FDA approved method; and/or - radiographic progression after the last line of systemic therapy. [0322] In certain embodiments, provided herein are methods for treating a subject having triple negative breast cancer (TNBC) and one or more (e.g., all) of the following characteristics: - a tumor having a PD-L1 score (CPS) less than 10 as measured by immunohistochemistry, for example, using Agilent/Dako’s PD-L1 immunohistochemistry 22C3 pharmDx assay; - histologically documented (metastatic or locally advanced) TNBC, including a HER2 status making the subject ineligible for trastuzumab as defined per the American College of Physicians (ACP) guidelines or equivalent, negative ER expression, and negative PR expression: o lack of HER2 amplification as determined by in situ hybridization (ratio of HER2 to CEP17 smaller than 2.0 or single probe average HER2 gene copy number smaller than 4 signals/cell), or HER2 expression level 0, 1+, or 2+ as measured by immunohistochemistry, and
o ER and PR negative (as defined as < 1% of cells expressing hormonal receptors via immunohistochemistry analysis); - has not previously received chemotherapy or targeted systemic therapy for inoperable locally advanced or metastatic TNBC; and/or - has disease measurable with at least one unidimensional measurable lesion by RECIST 1.1. [0323] In certain embodiments, provided herein are methods for treating a subject having a HER23+ tumor and one or more (e.g., all) of the following characteristics: - any solid tumor except breast cancer or gastric cancer and a history of erbb2 amplification within the tumor and one of the following 1) HER23+ scoring by immunohistochemistry documented in the subject’s most recent biopsy within 6 months, post radiographic progression on the last line of therapy or 2) HER23+ scoring by immunohistochemistry during the screening window; and - treatment with at least one line of an approved or established therapy. [0324] In certain embodiments, provided herein are methods for treating a subject having gastric cancer and one or more (e.g., all) of the following characteristics: - advanced (unresectable/recurrent/metastatic) gastric cancer or cancer of the gastro- esophageal junction as per the 7
th American Joint Committee on Cancer (AJCC) classification; - has a HER2 positive tumor per the American College of Physicians (ACP) guidelines, and therefore eligible for trastuzumab treatment; - previously received treatment with a first line of therapy including a platinum salt and a fluoropyridine in combination with trastuzumab or a biosimilar to trastuzumab; - has progressed after the first line therapy; - has received only one line of therapy for the treatment of metastatic disease; - has disease measurable with at least one unidimensional measurable lesion by RECIST 1.1; and/or - has a tumor that is not known to be microsatellite instability (MSI) high.
[0325] In certain embodiments, provide herein are methods of treating a subject having a HER23+ solid tumor and one or more (e.g., all) of the following characteristics: - has disease measurable with at least one unidimensional measurable lesion by RECIST 1.1; - has any solid tumor except breast cancer or gastric cancer and a history of erbb2 amplification within the tumor and one of the following 1) HER23+ scoring by immunohistochemistry documented in the subject’s most recent biopsy within 6 months, post radiographic progression on the last line of therapy, or 2) HER23+ scoring by immunohistochemistry during the screening window; and/or - has received treatment with at least one line of an approved or established therapy. [0326] In certain embodiments, provided herein are methods for treating a subject having a malignancy of epithelial origin and is eligible for treatment with nivolumab in accordance with its label. In certain embodiments, provided herein are methods for treating a subject having a metastatic breast cancer, and has failed to respond to a combination chemotherapy for metastatic disease or relapsed within 6 months of the adjuvant chemotherapy, did not have exposure to taxanes in the last 6 months, and is eligible for treatment with nab-paclitaxel in accordance with its label. [0327] In certain embodiments, provided herein are methods for treating cancer in a subject having treated in accordance with the methods disclosed herein does not meet one or more of the exclusion criteria described in Example 3. In certain embodiments, the subject treated in accordance with the methods disclosed herein does not meet any of the exclusion criteria described in Example 3. [0328] In certain embodiments, provide herein are methods of treating cancer in a subject who: - is not currently being treated with an immunotherapy, an immunosuppressive drug such as a chemotherapy or systemic corticosteroid (except for a short term treatment of allergic reactions or for the treatment of immunotherapy-related adverse event (irAE) with a systemic steroid, or treatment with a topical or inhalatory steroid with no or minimal systemic effect), or other experimental pharmaceutical product; - is not currently being treated with a tyrosine kinase inhibitor targeting HER2, or any recombinant molecule targeting HER2 or NKG2D;
- is not currently being treated with a growth factor such as granulocyte colony stimulating factor or granulocyte macrophage colony stimulating factor (except for erythropoietin and erythropoietin analogs); - is not currently being treated with bisphosphonate or denosumab unless treatment was initiated more than 14 days prior to receiving the first administration of multi-specific binding protein; - has not previously been treated with drugs that specifically target the HER2 pathway (except subjects treated with a monoclonal antibody and provided a four week washout period, or subjects treated with a tyrosine kinase inhibitor providing a two week washout period); - is not currently being treated with cytoreductive therapy, radiotherapy (except palliative bone directed radiotherapy), cytokine therapy (except for erythropoietin), major surgery (except diagnostic biopsy), or use of any investigational drug within 28 days prior to treatment with multi-specific binding protein; - has not previously had a malignant disease within the last three years other than the target malignancy to treated by the multi-specific binding protein (except basal or squamous cell carcinoma of the skin or cervical carcinoma in situ); - does not have rapidly progressive disease; - does not have active or a history of central nervous system (CNS) metastases; - has not previously received an organ transplant (including autologous or allogenic stem cell transplantation); - does not have significant acute or chronic infections (including a positive test for human immunodeficiency virus (HIV)), or active or latent hepatitis B or active hepatitis C; - does not have a pre-existing autoimmune disease (except subjects with vitiligo), needing treatment with systemic immunosuppressive agents for more than 28 days within the last three years or clinically relevant immunodeficiencies (e.g., dys- gammaglobulinemia or congenital immunodeficiencies), or fever within seven days; - does not have known severe hypersensitivity reactions to mAbs (≥ Grade 3 NCI- CTCAE v5.0), any history of anaphylaxis, or uncontrolled asthma (e.g., three or more features of partly controlled asthma);
- does not have persisting toxicity related to prior therapy > Grade 1 NCI-CTCAE v5.0 (except alopecia and sensory neuropathy ≤ Grade 2); - is not a pregnant or lactating female; - does not have known alcohol or drug abuse; and/or - does not have serious cardiac illness or medical conditions including, but not limited to: o a history of New York Heart Association class III or IV heart failure or systolic dysfunction (LVEF < 55%); o high-risk uncontrolled arrhythmias, i.e. tachycardia with a heart rate > 100/min at rest; o significant ventricular arrhythmia (ventricular tachycardia) or higher-grade AV-block (second degree AV-block Type 2 (Mobitz 2) or third-degree AV- block); o angina pectoris requiring anti-anginal medication; o clinically significant valvular heart disease; o evidence of transmural infarction on electrocardiogram (ECG); or o poorly controlled hypertension (defined by: systolic > 180 mm Hg or diastolic > 100 mm Hg). [0329] It is contemplated that the method disclosed herein of treating a patient having a cancer specified above can be performed with or without combination with a corticosteroid for reducing one or more infusion-related reactions. Monotherapies and Combinational Use with Other Cancer Therapies [0330] The multi-specific binding protein disclosed herein can be used as a monotherapy or in combination with one or more therapies. In certain embodiments, the multi-specific binding protein is used as a monotherapy in accordance with the dosage regimen disclosed herein. In other embodiments, the multi-specific binding protein is used in combination with one or more therapies administered in accordance with a dosage regimen known to be suitable for treating the particular subject with the particular cancer, and the multi-specific binding protein is administered in accordance with the dosage regimen
disclosed herein. In certain embodiments, the method of treatment disclosed herein is used as an adjunct to surgical removal of the primary lesion. [0331] Exemplary therapeutic agents that may be used in combination with the multi- specific binding protein include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma (IFN-γ), colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor and variations of the aforementioned agents that may exhibit differential binding to their cognate receptors, or increased or decreased serum half- life. [0332] An additional class of agents that may be used as part of a combination therapy in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T^lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the United States Food and Drug Administration for treating melanoma and may be used as part of a combination therapy described herein. [0333] Yet other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., herceptin) and non-cytotoxic agents (e.g., tyrosine-kinase inhibitors). [0334] Yet other categories of anti-cancer agents include, for example: (i) an inhibitor selected from an ALK Inhibitor, an ATR Inhibitor, an A2A Antagonist, a Base Excision Repair Inhibitor, a Bcr-Abl Tyrosine Kinase Inhibitor, a Bruton's Tyrosine Kinase Inhibitor, a CDC7 Inhibitor, a CHK1 Inhibitor, a Cyclin-Dependent Kinase Inhibitor, a DNA-PK Inhibitor, an Inhibitor of both DNA-PK and mTOR, a DNMT1 Inhibitor, a DNMT1 Inhibitor
plus 2-chloro-deoxyadenosine, an HDAC Inhibitor, a Hedgehog Signaling Pathway Inhibitor, an IDO Inhibitor, a JAK Inhibitor, a mTOR Inhibitor, a MEK Inhibitor, a MELK Inhibitor, a MTH1 Inhibitor, a PARP Inhibitor, a Phosphoinositide 3-Kinase Inhibitor, an Inhibitor of both PARP1 and DHODH, a Proteasome Inhibitor, a Topoisomerase-II Inhibitor, a Tyrosine Kinase Inhibitor, a VEGFR Inhibitor, and a WEE1 Inhibitor; (ii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF. [0335] In certain embodiments, the method of the present invention further includes administering to the subject an anti-PD-1 antibody. Many anti-PD-1 antibodies have been developed for therapeutic purposes and are described in, for example, Gong et al., (2018) J. ImmunoTher Cancer 6:8. [0336] In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, 0.1 to 10 mg/kg, 0.1 to 3 mg/kg, 0.1 to 1 mg/kg, 0.1 to 0.3 mg/kg, 0.3 to 10 mg/kg, 0.3 to 3 mg/kg, 0.3 to 1 mg/kg, 1 to 10 mg/kg, 1 to 3 mg/kg, or 3 to 10 mg/kg of nivolumab is administered to the subject. In certain embodiments, 120 to 600 mg, 120 to 480 mg, 120 to 360 mg, 120 to 240 mg, 240 to 600 mg, 240 to 480 mg, 240 to 360 mg, 360 to 600 mg, 360 to 480 mg, or 480 to 600 mg of nivolumab is administered to the subject. In certain embodiments, 120 mg, 240 mg, 360 mg, 480 mg, or 600 mg of nivolumab is administered to the subject. In certain embodiments, 480 mg of nivolumab is administered on day 1 of the initial treatment cycle. In certain embodiments, if the subject receives one or more subsequent treatment cycles, 480 mg of nivolumab is administered once every four weeks in the subsequent treatment cycles, starting from day 1 of each subsequent treatment cycle. In certain embodiments, nivolumab is administered as a 30 minute, 35 minute, 45 minute, 50 minute, 60 minute, 65 minute, 70 minute, 75 minute, 80 minute, 85 minute, or 90 minute intravenous infusion in four-week treatment cycles. [0337] In certain embodiments, a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion, and nivolumab is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion in four-week treatment cycles. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 min. intravenous infusion, and nivolumab is administered as a 30 min. intravenous infusion in four-week treatment cycles.
[0338] In certain embodiments, a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered in a first treatment cycle in combination with nivolumab, where the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation and nivolumab are both administered on day 1, and the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered alone at day 8. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered in combination with nivolumab in subsequent-week treatment cycles after an initial cycle, where the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation and nivolumab are both administered on day 1. [0339] In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, 100 to 300 mg, 110 to 300 mg, 120 to 300 mg, 130 to 300 mg, 140 to 300 mg, 150 to 300 mg, 160 to 300 mg, 170 to 300 mg, 180 to 300 mg. 190 to 300 mg, 200 to 300 mg, 210 to 300 mg, 220 to 300 mg, 230 to 300 mg, 240 to 300 mg, 250 to 300 mg, 260 to 300 mg, 270 to 300 mg, 280 to 300 mg, 290 to 300 mg, 100 to 280 mg, 110 to 280 mg, 120 to 280 mg, 130 to 280 mg, 140 to 280 mg, 150 to 280 mg, 160 to 280 mg, 170 to 280 mg, 180 to 280 mg. 190 to 280 mg, 200 to 280 mg, 210 to 280 mg, 220 to 280 mg, 230 to 280 mg, 240 to 280 mg, 250 to 280 mg, 260 to 280 mg, 270 to 280 mg, 100 to 260 mg, 110 to 260 mg, 120 to 260 mg, 130 to 260 mg, 140 to 260 mg, 150 to 260 mg, 160 to 260 mg, 170 to 260 mg, 180 to 260 mg. 190 to 260 mg, 200 to 260 mg, 210 to 260 mg, 220 to 260 mg, 230 to 260 mg, 240 to 260 mg, 250 to 260 mg, 100 to 240 mg, 110 to 240 mg, 120 to 240 mg, 130 to 240 mg, 140 to 240 mg, 150 to 240 mg, 160 to 240 mg, 170 to 240 mg, 180 to 240 mg. 190 to 240 mg, 200 to 240 mg, 210 to 240 mg, 220 to 240 mg, 230 to 240 mg, 100 to 220 mg, 110 to 220 mg, 120 to 220 mg, 130 to 220 mg, 140 to 220 mg, 150 to 220 mg, 160 to 220 mg, 170 to 220 mg, 180 to 220 mg. 190 to 220 mg, 200 to 220 mg, 210 to 220 mg, 100 to 200 mg, 110 to 200 mg, 120 to 200 mg, 130 to 200 mg, 140 to 200 mg, 150 to 200 mg, 160 to 200 mg, 170 to 200 mg, 180 to 200 mg, or 190 to 200 mg of pembrolizumab is administered to the subject. In certain embodiments, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg of pembrolizumab is administered to the subject. In certain embodiments, 200 mg of pembrolizumab is administered on Day 1 of the initial treatment cycle. In certain embodiments, if the subject receives one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles, starting from Day 1 of the first subsequent treatment cycle. In certain embodiments, pembrolizumab is administered as a 30 minute, 35
minute, 45 minute, 50 minute, 60 minute, 65 minute, 70 minute, 75 minute, 80 minute, 85 minute, or 90 minute intravenous infusion in three-week treatment cycles. [0340] In certain embodiments, a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion, and pembrolizumab is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion in three-week treatment cycles. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 min. intravenous infusion, and pembrolizumab is administered as a 30 min. intravenous infusion in three-week treatment cycles. [0341] In certain embodiments, a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered in a first treatment cycle in combination with pembrolizumab, where the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation and pembrolizumab are both administered on day 1, and the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered alone at day 8. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered in combination with pembrolizumab in subsequent 3-week treatment cycles after an initial cycle, where the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation and pembrolizumab are both administered on day 1. [0342] In certain embodiments, the method of the present invention further includes administering to the subject a cytoskeletal-disrupting chemotherapeutic agent. Many such agents have been developed for therapeutic purposes and are described in, for example, Ong et al., (2020) Cancers 12(1): 238. In certain embodiments, the cytoskeletal-disrupting chemotherapeutic agent is nab-paclitaxel. In certain embodiments, 50 to 300 mg/m
2, 50 to 200 mg/m
2, 50 to 150 mg/m
2, 50 to 100 mg/m
2, 100 to 300 mg/m
2, 100 to 200 mg/m
2, 100 to 150 mg/m
2, 150 to 300 mg/m
2, 150 to 200 mg/m
2, or 200 to 300 mg/m
2 of nab-paclitaxel is administered to the subject. In certain embodiments, 80 mg/m
2, 100 mg/m
2, 125 mg/m
2, 200 mg/m
2, or 260 mg/m
2 of nab-paclitaxel is administered to the subject. In certain embodiments, 100 mg/m
2 of nab-paclitaxel is administered on day 1, day 8, and day 15 of the initial treatment cycle. In certain embodiments, if the subject receives one or more subsequent treatment cycles, 100 mg/m
2 of nab-paclitaxel is administered three times every
four weeks in the subsequent treatment cycles, on day 1, day 8, and day 15 of each subsequent treatment cycle. In certain embodiments, nab-paclitaxel is administered as a 30 minute, 35 minute, 45 minute, 50 minute, 60 minute, 65 minute, 70 minute, 75 minute, 80 minute, 85 minute, or 90 minute intravenous infusion in four-week treatment cycles. [0343] In certain embodiments, a multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion, and nab-paclitaxel is administered as a 30 min., 45 min., 60 min., 75 min., or 90 min. intravenous infusion in four-week treatment cycles. In certain embodiments, the multi-specific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 min. intravenous infusion, and nab-paclitaxel is administered as a 30 min. intravenous infusion in four-week treatment cycles. Therapeutic Outcome [0344] In certain embodiments, the method of treatment disclosed herein results in a disease response or improved survival of the subject or patient. For example, in certain embodiments, the disease response is a complete response, a partial response, or a stable disease. In certain embodiments, the improved survival is improved progression-free survival (PFS) or overall survival. Improvement (e.g., in PFS) can be determined relative to a period prior to initiation of the treatment of the present disclosure. Methods of determining disease response (e.g., complete response, partial response, or stable disease) and patient survival (e.g., PFS, overall survival) for BTC (e.g., advanced BTC, metastatic BTC), or biliary tract tumor therapy, are routine in the art and are contemplated herein. In some embodiments, disease response is evaluated according to RECIST 1.1 after subjecting the treated patient to contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) of the affected area (e.g., chest/abdomen and pelvis covering the area from the superior extent of the thoracic inlet to the symphysis pubis). EXAMPLES [0345] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.
Example 1: Formulation, Packaging, and Storage of A49-F3’-TriNKET-Trastuzumab [0346] The formulations listed in Table 12 were evaluated, in duplicate and randomized, to assess the effects of the pH and excipients on the stability of A49-F3’- TriNKET-Trastuzumab (Kermit BDS lot 7443-C3, 11.9 mg/mL). A49-F3’-TriNKET- Trastuzumab underwent buffer exchange into the respective buffer and excipient combinations by centrifugal ultrafiltration (Amicon Ultra-430k devices MWCO) to a target concentration of 30 mg/mL. Following the final buffer exchange and confirmation of the target concentration, each formulated sample was filter sterilized using a 0.22 μm EMD Millipore Ultrafree – CL centrifugal filter device with Durapore membrane (Fisher Scientific Cat. # UFC40GVOS). Following sterile filtration, each formulation was handled aseptically in a laminar flow hood. The formulated samples were spiked with polysorbate 80 (PS80) to a final concentration of 0.01%. An aliquot of each formulation was removed for time zero testing, and the remaining material was split into two equal sized aliquots into depyrogenated Type 1 borosilicate glass vials, 2 mL x 13 mm (West Pharmaceuticals Cat. # 68000377), stoppered with 13 mm Fluorotec stoppers (West Pharmaceuticals Cat. # 19700302), and sealed. The time zero aliquots were used for the initial time point testing per Table 13. One vial was stored at 2 – 8 °C and the other vial was placed at 50 °C for a 3-week accelerated stability study. Following the 3-week incubation, the 2 – 8 °C and 50 °C samples were analyzed according to the test methods indicated in Table 13.